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ANIMAL   MICROLOGY 


THE  UNIVERSITY  OF  CHICAGO  PRESS 
CHICAGO.  ILLINOIS 

Bgenta 
THE  BAKER  &  TAYLOR  COMPANY 

NEW  YORK 

CAMBRIDGE  UNIVERSITY  PRESS 

LONDON    AND   EDINBURGH 


ANIMAL    MICROLOGY 

PRACTICAL  EXERCISES  IN 
MICROSCOPICAL  METHODS 


BY 

MICHAEL  F.  GUYER,  Ph.D. 

Professor  of  Zodlogy  in  zhe  University  of  Cincinnati 


B 


•  ■"       -.1 


THE  UNIVERSITY  OF  CHICAGO  PRESS 
CHICAGO,  ILLINOIS 

K\4 


Copyright  1906  By 
The  University  of  Chicago 


All  rights  reserved 


Published  November  1906 
Second  Impression  November  1910 


Composed  and  Printed  By 

The  University  of  Chicago  Press 

Chicago,  Illinois,  U.S.A. 


PREFACE 

For  the  past  ten  years  it  has  been  a  part  of  the  writer's  duties 
to  give  instruction  in  microscopical  technique,  and  it  has  seemed 
to  him  that  there  is  need  for  a  series  of  practical  exercises  which 
will  serve  to  guide  the  beginner  through  the  maze  of  present-day 
methods,  with  the  greatest  economy  of  time,  by  drilling  him  in  a 
few  which  are  thoroughly  fundamental  and  standard.  The  book 
is  intended  primarily  for  the  beginner  and  gives  more  attention 
to  the  details  of  procedure  than  to  discriminations  between 
reagents  or  the  review  of  special  processes.  The  student  is  told 
what  to  do  with  his  material,  step  by  step,  and  why  he  does  it; 
at  what  stages  he  is  likely  to  encounter  difficulties  and  how  to 
avoid  them;  if  his  preparation  is  defective,  what  the  probable 
cause  is  and  the  remedy.  In  short,  the  book  attempts  to  famil- 
iarize the  student  with  the  little  "tricks"  of  technique  which 
are  commonly  left  out  of  books  on  methods  but  which  mean 
everything  in  securing  good  results. 

A  very  brief,  non-technical  account  of  the  principles 
of  the  microscope  is  inserted  (Appendix  A)  with  the 
idea  of  giving  the  student  just  enough  of  the  theoretical  side  of 
microscopy  to  enable  him  to'  get  satisfactory  results  from  his 
microscope.  The  microscope  is  so  ably  treated  in  the  excellent 
works  of  Gage  [The Microscope)  and  Carpenter  [The  Microscope 
and  Its  Revelations)  that  the  writer  feels  himself  absolved  from 
any  further  responsibility  in  this  matter. 

The  aim  of  the  entire  book  is  to  be  practical:  to  omit  every- 
thing that  is  not  essential;  and,  above  all,  to  give  definite  state* 
ments  about  things.  Appended  to  each  chapter  is  a  series  of 
memoranda  which  serve  to  supply  additional  information  that  is 
more  or  less  pertinent  without  obscuring  the  main  features  of  the 
method  under  consideration. 

In  Appendix  B  the  formulae  for  a  number  of  the  most  widely 
used  reagents  are  given  with  comments  upon  their  uses  and  manipu- 
lation.    Following  this  (Appendix  C)  is  a  concise  table  of  a  large 


543, 


vi  Animal  Micrology 

number  of  tissues  and  organs  with  directions  for  properly 
preparing  them  for  microscopical  study. 

Inasmuch  as  every  experienced  worker  has  his  own  "best" 
method  for  the  preparation  of  almost  any  tissue,  it  is  manifestly 
impossible  to  give  all  "best  methods"  in  such  a  table.  The  writer 
believes,  however,  that  the  student  will  find  the  methods 
recommended  all  good  ones  which  will  yield  satisfactory  results. 

In  Appendix  D  some  directions  are  given  for  collecting  and 
preparing  material  for  an  elementary  course  in  zoology. 

It  is  hoped  that  the  volume  will  prove  of  use:  (1)  as  a  class 
textbook;  (2)  as  a  guide  to  the  independent  individual  worker 
(teacher,  physician,  college  or  medical  student,  or  novice)  ;  (3)  as 
a  reference  book  for  teachers,  in  the  preparation  of  material  for 
courses  in  elementary  zoology,  histology,  or  embryology. 

In  the  matter  of  expressing  his  obligations  the  writer  is  at  a 
loss  to  know  just  what  to  do.  Many  of  the  methods  in  microscopi- 
cal technique  have  been  handed  down  tradition- wise  from  one 
worker  to  another  until  their  origin  is  unknown ;  they  are  the 
accumulated  experiences  of  several  generations  of  workers.  Fur- 
thermore, many  points  have  been  absorbed,  as  it  were,  by  the 
writer,  from  fellow- workers  in  the  Universities  of  Chicago, 
Nebraska,  and  Cincinnati,  respectively;  consequently  the  obliga- 
tion cannot  be  specifically  expressed.  Where  the  name  of  the 
originator  of  a  method  is  known,  due  credit  has  been  given.  The 
books  to  which  the  author  is  most  heavily  indebted  are  the  vol- 
umes of  Gage,  and  Carpenter,  already  mentioned,  Lee's  Microto- 
misfs  Vade-Mecum,  Whitman's  Methods  in  Microscopical  Anat- 
omy and  Embryology,  Hardesty's  Neurological  Technique,  Fos- 
ter and  Balfour's  Elements  of  Embryology,  Minot's  Laboratory 
Text-book  of  Embryology,  Huber's  translation  of  the  Bohm- 
Davidoff  Text-book  of  Histology,  Stohr's  Text-book  of  Histology, 
Mallory  and  Wright's  Pathological  Technique,  Bausch's  Manipu- 
lation of  the  Microscope,  and  the  Journal  of  Applied  Micros- 
copy. Grateful  acknowledgment  is  also  made  to  the  various 
manufacturers  of  microscopical  instruments  and  appliances  for 
the  loan  of  most  of  the  cuts  which  have  been  used  in  this  volume. 

M.  F.  G. 


CONTENTS 

PA0B 

Introductory .        1 

Apparatus  and  Supplies  Required,  1;  General  Rules,  5. 

Chapter  I.     Preparation  of  Reagents 7 

Practical  Exercise,  7;  Memoranda,  13. 

.Chapter  II.    General  Statement  of  Methods 15 

Killing,  Fixing  and  Hardening,  16;  Washing,  17;  Dehydrating,  18; 
Preserving,  18;  Staining,  19;  Clearing,  21;  Mounting,  22;  Imbedding, 
22;  Affixing  Sections,  23;  Decolorizing,  24;  Bleaching,  24;  Corrosion, 
24;  Decalcification  and  Desilicidation,  24;  Injection  25;  Isolation  of 
Histological  Elements,  25;  Normal  or  Indifferent  Fluids,  25;  Gen- 
eral Scheme  for  Mounting  Whole  Objects  (In  Toto  Preparations)  or 
Sections,  26. 

Chapter  III.    Killing  and  Fixing 27 

Cautions,  27;  Alcohol  Fixation,  28;  Fixing  with  Gilson's  Mercuro- 
Nitric  Mixture.  28;  Fixing  with  Erlicki's  Fluid,  29;  Formalin  as  a 
Fixing  Reagent,  29;  Memoranda.  30. 

Chapter  IV.     Simple  Section  Methods 33 

Free-Hand  Section  Cutting,  33;   Memoranda,  34. 

Chapter  V.     The  Paraffin  Method;   Imbedding  and  Sectioning     .      37 
The  Method,  37;  Memoranda,  43;  Tabular  Statement  of  Difficulties 
Likely  to  Be  Encountered  in  Sectioning  in  Paraffin  and  the  Probable 
Remedy,  46. 

Chapter  VI.  The  Paraffin  Method:  Staining  and  Mounting  .  .  49 
Staining  with  Hematoxylin,  49;  Double  Staining  in  Hematoxylin 
and  Eosin,  51;  Double  Staining  in  Carmine  and  Lyons  Blue,  51; 
Staining  with  Heidenhain's  Iron-Alum  Hematoxylin,  52;  Bordeaux 
Red  and  Iron-Hematoxylin,  53;  Staining  in  Bulk  before  Section- 
ing, 54;  Paraffin  Method  for  Delicate  Objects,  54;  Memoranda,  55. 

Chapter  VII.     The  Celloidin  Method 59 

The  Method,  59;  Staining  Celloidin  Sections  in  Hematoxylin  and 
Eosin,  62;   Memoranda,  62. 

Chapter  VIII.     The  Freezing  Method      .     .     .     ......     .       67 

The  Method,  67;  Memoranda,  69. 

Chapter  IX.     Metallic  Substances  for  Color  Differentiation    .       71 
A  Golgi  Method  for  Nerve  Cells  and  Their  Ramifications,  71;  Memo- 
randa, 72;    Silver  Nitrate  Method  for  Nerve,  73;    Gold  Chloride 
Method  for  Nerve  Endings,  74. 

vii 


viii  Animal  Micrology 

Chapter  X.     Isolation  of  Histological  Elements.    Minute  Dis- 
sections      ' 75 

Dissociation  by  Means  of  Formaldehyde,  75;   Isolation  of  Muscle 
Fibers  by  Maceration  and    Teasing,  75;  Maceration  by  Means  of 
Hertwig's  Fluid,  76;  Minute  Dissection  and  Mounting  of  Various 
Parts  of  Insects,  77;  Memoranda,  78. 

Chapter  XI.     Tooth,  Bone,  and  Other  Hard  Objects 79 

Sectioning  Decalcified  Tooth,  79;  Sectioning  Decalcified  Bone,  79; 
Sectioning  Bone  by  Grinding,  80;  Memoranda,  80. 

Chapter  XII.     Injection  of  Blood  and  Lymph  Vessels      ....       81 
Red  Injection  Mass,  81;  Blue  Injection  Mass,  81;   Injection  with  a 
Syringe,  81;  Memoranda,  84. 

Chapter   XIII.    Objects    of    General   Interest:    Cell-Making, 

Fluid  Mounts,  "In  Toto"  Preparations,  etc 87 

Turning  Cells,  87;  Mounting  in  Glycerin  (Water  Mites,  Transparent 
Larvae),  88;  Killing  and  Mounting  Hydra,  89;  Mounting  in  Glycerin 
Jelly  (Small  Crustacea,  etc.),  89;  Mounting  in  Balsam  (Flat  Worms, 
Mosquito,  Gnat,  Aphid),  90;  Opaque  Mounts  (Beetles,  Wings  of 
Moths  and  Butterflies,  Head  of  Fly,  Foreleg  of  Dytiscus),  92; 
Memoranda  (Including  Directions  for  Mounting  Other  Forms),  93. 

Chapter  XIV.    Blood 97 

Examination  of  Fresh  Blood,  97;  Effects  of  Reagents,  97;  To  Demon- 
strate Blood  Platelets,  97;  Stained  Preparation  of  Fibrin,  97;  Crystals 
of  Blood,  97;  Cover-Glass  Preparations  (Dry),  98;  Rapid  Method,  99; 
Enumeration  of  Blood  Corpuscles,  99;  Observation  of  the  Blood 
Current,  101;  Inflammation,  101;  Memoranda,  101. 

Chapter  XV.    Bacteria 105 

Bacterial  Examination,  105;  Cover-Glass  Preparation  from  Fluid 
Media,  105;  Staining  and  Mounting,  106;  Bacteria  in  Tissues,  107; 
Methylen  Blue  Stain  for  Bacteria  in  Tissues,  107;  Gram's  Method  for 
Bacteria  in  Tissues,  107;  Hanging-Drop  Preparations,  108;  Memo- 
randa, 108. 

Chapter  XVI.    Some  Embryological  Methods:   The  Chick;   Sec- 
tions and  Whole  Mounts;  Other  Forms 113 

The  Chick,  113;  Series  of  Chick  Embryos  for  a  Course  in  Embry- 
ology, 115;  Measuring  the  Length  of  Embryos,  116;  Orientation  of 
Young  Chick  Embryos,  116;  Preparation  of  Material  for  the  Embry- 
ology of  Teleosts,  117;  Preparation  of  Material  for  the  Embryology 
of  Amphibia,  118;  Early  Stages  of  the  Mammalian  Embryo,  119; 
Older  Stages  of  the  Mammalian  Embryo,  120;  Maturation,  Fertiliza- 
tion, and  Segmentation  in  Mammals,  121;  Artificial  Fecundation 
(Echinoderms,  Amphibia,  Teleosts),  122;  Early  Cleavage  in  Living 
Material,  123;*  Polar  Bodies,  Fertilization  and  Early  Cleavage  in 


ConteMs  ix 

Ascaris  (Sections),  123;  Directions  for  Orienting  Serial  Sections,  123; 
Orientation  of  Objects  in  the  Imbedding  Mass,  124;  Human  Em- 
bryos, 126. 

Chapter  XVII.     Reconstruction  of  Objects  from  Sections    .     .     .     127 
Reconstruction  in  Wax,  127;  Geometrical  Reconstruction,  129. 

Appendix  A.  The  Microscope  and  Its  Optical  Principles  .  .  .  133 
Optical  Principles,  133;  Images,  135;  The  Simple  Microscope,  136; 
The  Compound  Microscope,  137;  Defects  in  the  Image,  138;  Nomen- 
clature or  Rating  of  Objectives  and  Oculars,  144;  Some  Common 
Microscopical  Terms  and  Appliances  (Alphabetically  Arranged),  145; 
Manipulation  of  the  Compound  Microscope,  157. 

Appendix  B.    Some  Standard  Reagents  and  Their  Uses    .     .    .    .     161 
Fixing  and  Hardening  Agents,  161;  Stains,  170;  Normal  or  Indif- 
ferent Fluids,  187;  Dissociating  Fluids,  187;  Decalcifying  Fluids,  189. 

Appendix  C.  Table  of  Tissues  and  Organs  with  Methods  of  Prepa- 
ration         ......     190 

Appendix  D.     Preparation  of  Microscopic  Material  for  a  Course 

in  General  Zoology , ,    215 

Appendix  E.    Table  of  Equivalent  Weights  and  Measures  .     .     .    227 


INTRODUCTORY 

APPARATUS  AND  SUPPLIES  REQUIRED 

The  student  should  provide  himself  with  the  following  sup- 
plies : 

One  half -gross  box  best  grade  glass  slides,  standard  size  (25x75  mm.). 
One-half  ounce,  18  mm.  or  §  in.,  round  cover-glasses,  medium  thickness, 

(o.  18  mm.). 
Thirty,  25x50  mm.  cover-glasses,  medium  thickness. 
Two  or  three  Pillsbury  slide  boxes  (Fig.  1). 
One  box  of  labels  for  slides. 
Three  to  six  camel's  hair  brushes  (Fig.  2). 
Six  pipettes  (Fig.  3). 
One  set  of  dissecting  instruments  as  follows: 

One  large  scalpel  or  cartilage  knife  (Fig.  4). 

One  small  scalpel  (Fig.  5). 

Two  needles  (Fig.  6). 

One  fine  straight  scissors  (Fig.  7). 

One  fine  straight  dissecting  forceps,  file-cut  points  (Fig.  8). 

One  blow-pipe  (Fig.  9). 

One  section  lifter  (Fig.  10). 
To  which  may  well  be  added : 

One  heavy  scissors  (Fig.  11). 

One  curved  scissors  (Fig.  12). 

One  heavy  forceps  (Fig.  13). 

One  fine  forceps,  curved  tips  (Fig.  14). 
One  horn  spoon. 

One  desk  memorandum  calendar. 
Blank  cards  (about  75x100  mm.)  for  keeping  records  of  experiments. 

The  kind  of  card  used  for  library  card  catalogue  will  do. 
One  section  razor  (Fig.  15). 
A  piece  of  moderately  heavy  copper  wire  with  one  end  hammered  out  to 

a  width  of  7  to  10  mm. 
Towels. 

A  glass-marking  pencil  (wax)  or  writing  diamond  will  be  found  useful. 
See,  however,  Memorandum  21,  chap.  vi. 

1 


Animal  Micrology 


Fig.  1 


Fig.  7 


Apparatus  and  Supplies  Required 


Fig.  17 


Fig.  12  Fig.  13  Fig.  14 


Fig.  18 


^0 

Fig.  19  Fig.  20 


Fig.  21 


Fig.  22 


Fig.  23 


4  Animal  Micrology 

Apparatus  ordinarily  supplied  by  the  laboratory: 

Desk  with  drawers. 

Locker  for  microscope. 

Compound  microscope  and  accessories  (Appendix  A). 

Dissecting  microscope  (Fig.  66). 

Microtomes  (Figs.  27,  28, 29,  32,  33). 

Paraffin  oven  (Figs.  24,  25,  26). 

Tall  stenders  (about  85  mm.  deep).     Each  student  should  have  at  least 

eight  (Fig.  16). 
Coplin  staining  jars  (Fig.   17).    Tall  stenders   may  be  used  instead. 

About  eight  are  needed  for  each  student. 
Flat  stenders  (Fig.  18);  half  a  dozen  for  each  student. 
Syracuse  watch-glasses  (Fig.  19);  eight  to  each  student 
Balsam  bottle  (Fig.  20). 

Graduated  cylinders  for  measuring  liquids  (Fig.  21). 
Wash-bottle  (Fig.  22). 
Celloidin  bottle  (Fig.  23). 
Turntable  (Fig.  36). 
Injecting  apparatus  (Fig.  35). 
Reagent  bottles  and  vials. 

Other  apparatus  and  supplies  such  as  bone-forceps,  bone-saws,  glass 
tubing,  glass  rods,  beakers,  burners,  filter  paper,  funnels,  evapo- 
rating dishes,  sand  bath,  dropping-bottles,  balances,  mortar  and 
pestle,  etc. 
For  apparatus  or  supplies  not  listed  in  this  book  the  student  is  referred 
to  the  illustrated  catalogues  of  dealers  and  manufacturers  such  as:    The 
Bausch  and  Lomb  Optical  Co.,  Rochester,  N.  Y.;  The  Ernst  Leitz  Opti- 
cal Works,  Wetzlar,  Germany  (American  branch,  30  E.  18th  St.,  New 
York  City;  or,  32  Clark  St.,  Chicago);  The  Spencer  Lens  Co.,  Buffalo, 
N.  Y.;  Carl  Zeiss  Optical  Works,  Jena,  Germany;  R.  &  J.  Beck,  68,  Corn- 
hill,  London;  The  Kny-Scherer  Co.,  New  York  City;  Eimer  and  Amend, 
New  York  City;  Whitall,  Tatum  and  Co.  (especially  for  glassware),  New 
York  City. 


IMPORTANT  GENERAL  RULES 

1.  Keep  everything  clean! 

2.  Have  a  definite  place  in  your  desk  for  each  piece  of  appa- 
ratus and  arrange  reagents  in  order  on  top  of  it. 

3.  Use  cards  for  keeping  records  of  materials.  Each  card 
should  have  a  number  corresponding  to  that  of  each  special  object 
or  piece  of  tissue,  and  should  show  the  name  of  the  preparation, 
date,  reagents  used,  time  left  in  each  reagent,  in  short,  all  data 
concerning  the  manipulation  of  the  material. 

4.  Jot  down  in  a  blank  calendar  the  various  things  to  be  done 
at  future  dates,  such  as  changing  of  reagent  on  tissues,  etc.,  and 
then  go  over  this  memorandum  carefully  each  day  when  you  first 
come  into  the  laboratory. 

5.  Use  only  clean  vessels  in  preparing  reagents,  and  clean  up 
all  glassware  while  it  is  yet  moist. 

6.  Reserve  and  mark  a  separate  pipette  for  each  of  the  chief 
reagents  (absolute  alcohol,  oils,  acids,  etc.). 

7.  In  making  up  solutions,  1  gram  of  a  salt  in  100  c.c.  of 
liquid  is  reckoned  ordinarily  as  a  1  per  cent,  solution,  3  grams  as  a 
3  per  cent,  solution,  etc.  A  saturated  solution  contains  all  of  a 
given  substance  that  the  liquid  will  take  up.  When  a  solution  is 
called  for  without  specifying  the  solvent  an  aqueous  solution  is 
meant. 

8.  In  weighing  salts,  always  first  put  paper  in  the  scale  pans 
to  protect  them. 

9.  In  making  solutions  or  mixtures  in  which  only  a  small 
amount  of  one  reagent  is  used,  after  mixing,  pour  back  some  of 
the  mixture  into  the  small  vessel  and  rinse  it  thoroughly  in  order 
to  get  all  of  the  original  contents  out. 

10.  When  pouring  liquids  from  bottles  keep  the  label  of  the 
bottle  turned  toward  the  palm  of  the  hand.  Bo  not  lay  down 
stoppers  but  hold  them  by  their  tops  between  the  knuckles. 

11.  Before  leaving  the  laboratory  put  away  your  instruments 
and  clean  and  put  in  its  place  whatever  laboratory  apparatus  you 
may  have  been  using. 


CHAPTER  I 
PREPARATION  OF  REAGENTS 

The  following  reagents  should  be  prepared  by  each  student. 

1.  Grades  of  Alcohol. — To  obtain  a  given  per  cent,  of  alcohol 
through  dilution  of  a  higher  per  cent,  with  distilled  water,  sub- 
tract the  per  cent,  required  from  the  per  cent,  of  the  alcohol  to 
be  diluted ;  the  difference  is  the  proportion  of  water  that  must  be 
added.  Thus,  if  35  is  the  per  cent,  required,  and  95  the  per  cent, 
to  be  diluted,  then  95 — 35=60;  hence,  60  parts  of  water  and  35 
parts  of  95  per  cent,  alcohol  are  the  proportions  for  mixing. 

This  means  that  in  practice  one  needs  only  to  fill  the  graduated 
measuring  cylinder  to  the  same  number  as  the  per  cent,  required 
(e.  g.,  35)  with  the  alcohol  to  be  diluted  (e.  g.,  95)  and  then  fill 
up  to  the  per  cent,  of  the  latter  with  distilled  water.  In  this  way 
one  would  obtain  95  c.c.  of  alcohol  of  the  per  cent,  required,  if 
the  measuring  cylinder  is  graduated  in  cubic  centimeters. 

Prepare  about  250  c.c.  of  35,  50,  70,  and  83  per  cent,  alcohols 
respectively,  from  95  per  cent,  alcohol  and  distilled  water.  The 
commercial  alcohol  used,  though  really  about  96  per  cent.,  may 
be  figured  on  the  basis  of  95  per  cent. 

Owing  to  the  differences  in  the  specific  gravities  of  the  differ- 
ent percentages  of  alcohol,  the  above  method  gives  only  approxi- 
mate results;  they  are  sufficiently  accurate,  however,  for  most 
biological  work. 

2.  Absolute  Alcohol. — It  is  customary  in  most  laboratories  to 
purchase  so-called  absolute  alcohol  specially  prepared  for  labora- 
tory purposes.  Squibb's  absolute  alcohol  (99.8  per  cent.)  is 
commonly  used.  Inasmuch  as  such  alcohol  is  an  expensive 
reagent,  economy  sometimes  necessitates  that  the  student  under- 
take the  more  tedious  process  of  making  his  own  absolute  alcohol. 
Crystals  of  copper  sulphate  are  heated  until  the  water  of  crystal- 
lization is  driven  off  and  the  sulphate  is  left  as  a  white  powder. 
Such   anhydrous   sulphate    is  added  to  a    bottle  of  commercial 

7 


8  Animal  Micrology 

(96  per  cent.)  alcohol.  The  water  in  the  alcohol  immediately 
unites  with  it,  turning  it  blue.  Anhydrous  sulphate  should  be 
added  until  it  no  longer  turns  blue.  The  alcohol  is  then  filtered 
into  a  clean,  dry  bottle  which  must  have  a  tight-fitting  cork  or 
ground-glass  stopper.  It  is  well  to  smear  the  glass  stopper  with 
vaselin,  so  that  when  placed  in  the  bottle,  all  moisture  from  the 
air  may  be  completely  excluded. 

3.  Acid  Alcohol. — 

Alcohol  (70  per  cent.) 99  c.c. 

Hydrochloric  acid  (pure) 1  c.c. 

For  sections  use  the  mixture  only  a  few  seconds  or  minutes. 
For  material  stained  in  bulk,  add  twice  as  much  70  per  cent, 
alcohol  and  leave  the  object  in  it  until  sufficiently  decolorized 
(2  to  24  hours). 

4.  Ether  and  Alcohol. — Absolute  alcohol  and  sulphuric  ether 
equal  parts.  Quantity,  250  c.c.  Keep  the  ether  distant  from 
all  flames. 

5.  Normal  Saline. — Prepare  a  0.7  per  cent,  solution  of  sodium 
chloride  in  distilled  water.  This  is  termed  a  normal  salt  solution 
because  it  is  a  solution  of  about  the  same  density  as  natural 
lymph,  and  is  much  less  harmful  to  living  tissues  than  is  distilled 
water.     Quantity,  500  c.c. 

6.  Formalin  (also  termed  formal,  formol,  formolose). — Com- 
mercial formalin  is  a  40  per  cent,  solution  of  formaldehyde  in 
water.  A  4  per  cent,  solution  of  formalin  would  be  made  by 
taking  4  volumes  of  commercial  formalin  and  96  volumes  of 
water.  This  is,  however,  only  a  1.6  per  cent,  solution  of  formal- 
dehyde. Make  a  10  per  cent,  solution  of  formalin.  Quantity, 
250  c.c. 

7.  Gilson's  Mercuro-Nitric  Fixing  Fluid. — 

Bichloride  of  mercury  (corrosive  sublimate)       5  grams 
Nitric  acid  (approx.  80  per  cent.)     ....       4  c.c. 

Glacial  acetic  acid 1  c.c. 

Alcohol  (70  per  cent.) 25  c.c. 

Distilled  water 220  c.c. 

Quantity,  250  c.c. 


Chapter  I :   Preparation  of  Reagents  9 

Caution. — In  handling  corrosive  sublimate  do  not  use  metal 
instruments  because  it  corrodes  metal.  Use  a  glass  or  horn 
spatula. 

8.  Erlicki's  Solution. — 

Bichromate  of  potash 5  grams 

Sulphate  of  copper 2  grams 

Distilled  water 220  c.c. 

Pulverize  the  crystals  before  adding  the  water. 

9.  Gage's  Carbol-Xylol  Clearer. — 

Carbolic  acid  crystals  (melted) 25  parts 

Xylol 75  parts 

If  the  carbolic  acid  does  not  dissolve  in  the  xylol,  increase  the 

amount  of  the  latter.    Handle  the  acid  with  great  care.    Quantity, 

250  c.c. 

10.  Gage's  Formaldehyde  Dissociator. — 

Formalin,  commercial 0.5  c.c. 

Normal  saline 250.0  c.c. 

11.  Decalcifying  Solution. — 

Nitric  acid  (strong) 10  c.c. 

Alcohol  (70  per  cent.) 90  c.c. 

12.  Borax  Carmine  Stain   (Grenadier's). — 

Borax  (4  per  cent,  aqueous  solution)    ....   100  c.c. 
Carmine 1  gram 

Heat  until  the  carmine  dissolves,  then  add  100  c.c.  of  70  per 
cent,  alcohol.     Filter  after  24  hours. 

13.  Delafield's  Hematoxylin. — Prepare  100  c.c.  of  a  saturated 
aqueous  solution  of  ammonia  alum.  Dissolve  1  gram  of  hema- 
toxylin crystals  in  10  c.c.  of  absolute  alcohol,  and  add  it,  drop 
by  drop,  to  the  first  solution.  Expose  this  mixture  to  air  and 
light  for  several  weeks  (two  months  is  not  too  long)  to  "ripen." 
(Ripening  consists  in  an  oxidation  of  the  hematoxylin  to  form 
hematein.  This  may  be  accomplished  at  once  with  some  degree 
of  success  through  the  addition  of  a  few  cubic  centimeters  of  a 
neutralized  solution  of  peroxide  of  hydrogen.)  When  ripe,  filter 
the  solution  and  add  25  c.c.  of  glycerin,  and  25  c.c.  of  methyl 
alcohol  (see  memorandum  1).      It  is  well  to  have  a  stock  solution 


10  Animal  Micrology 

of  this   stain  already  prepared  to  be  used  in  case  the  student's 
preparation  is  not  ready  in  time 

Note. — At  this  point  the  student  should  begin  chapter  Hi 
in  order  that  no  time  may  be  lost.  The  present  chapter  may 
then  be  completed  while  the  tissues  are  becoming  fixed  and 
hardened. 

14.  Picro-Carmine. — This  stain  is  perhaps  best  procured 
ready  made  from  dealers.  If  it  is  desired  to  make  it,  consult 
Appendix  B,  reagent  63.     Quantity  necessary,  about  50  c.c. 

15.  Bordeaux  Red. — 

Bordeaux  red 1.0  gram 

Distilled  water 100.0  c.c. 

16.  Lyons  Blue. — 

Absolute  alcohol 100.0  c.c. 

Bleu  de  Lyon 0.3  gram 

17.  Eosin.— 

Eosin 0.5  gram 

Alcohol,  95  per  cent 100.0  c.c. 

18.  Iron  Hematoxylin  (Heidenhain's). — Two  solutions  are 
used.     They  are  not  to  be  mixed. 

Solution  I. 

Ferric  alum  (clear  violet  crystals)  ....       2.5  gram 
Distilled  water 100.0  c.c. 

Solution  II. 

Hematoxylin 0.5  gram 

Distilled  water 100.0  c.c. 

The  hematoxylin  should  ripen  (see  13)  for  some  three  or 
four  weeks. 

19.  Safranin. — 

Safranin  "O" .       1.0  gram 

Absolute  alcohol 10.0  c.c. 

Anilin  water 90.0  c.c. 

Make  the  "anilin  water"  by  shaking  up  5  c.c.  of  anilin  oil  in 
90  c.c.  of  distilled  water.  Filter  through  a  wet  filter.  Dis- 
solve the  safranin  in  the  anilin  water,  then  add  the  alcohol. 


Chapter  I:   Preparation  of  Reagents 


11 


20.  Canada  Balsam. — Dry  2  grams  of  Canada  balsam  on  a 
sand  bath,  or  in  a  warm  chamber  until  it  becomes  hard  (1  to  2 
hours  at  65  °C).  Do  not  overheat.  When  cool  add  enough  xylol 
to  make  a  thin,  syrupy  fluid.  Koll  a  sheet  of  paper  into  a  cone  to 
serve  as  a  funnel,  and  filter  the  fluid  through  absorbent  cotton. 


Fig.  24.— The  Lillie  Water-Bath. 

The  bath  consists  of  a  large  chamber  containing  a  series  of  drawers  of  equal  size, 
250 mm.  long,  100mm.  wide,  80  mm.  deep.  Each  drawer  has  copper  front  and  bottom  ;  the 
sides  and  back  are  perforated  zinc,  thus  securing  free  circulation  of  warm  air.  The  drawers 
are  separated  by  perforated  cross  partitions  and  run  on  slides  free  from  the  lateral  supports, 
thus  permitting  sufficient  circulation  of  warm  air  to  secure  equal  temperature  in  the  top 
and  bottom  of  the  bath.  Water  gauge  and  tubulatures  for  gas  regulator  and  thermometer 
are  provided.  This  bath  is  especially  adapted  to  class  work,  since  each  student  may  carry 
on  his  work  in  a  separate  drawer. 

Thicken  the  solution  slightly  by  leaving  the  cap  off  the  bottle  in 
a  place  free  from  dust,  and  allowing  some  of  the  xylol  to  evaporate. 
Or,  fill  your  balsam  bottle  one-third  full  of  the  liquid  xylol- 
balsam  now  on  the  market,  and  dilute  to  the  proper  consistency. 


12 


Animal  Micrology 


21.  Mayers  Albumen    Fixative. — Chop  the  white  of  an  egg 
with  scissors  and  filter  it  through  moist  filter  paper.     It  filters 

through  very  slowly.  Add  an 
equal  volume  of  glycerin,  and  a 
bit  of  salycilate  of  soda  (1  gram 
to  50  c.c.)  or  thymol  to  prevent 
putrefaction. 

22.  Celloidin. — Put  5  grams  of 
Schering's  shredded  or  granular 
celloidin  into  a  celloidin  bottle  (a 
bottle  with  glass  stopper  and 
ground  glass  cap)  and  dissolve  it 
in  equal  parts  of  absolute  alcohol 
and  ether  (see  4).  Add  only  suf- 
ficient fluid  (about  100  c.c.)  to 
make  a  thick,  syrupy  mass.  In  a 
second  celloidin  bottle  make  a  thin 
solution  by  taking  about  one-third 
of  the  original  solution  and  dilut- 
of    the    ether-alcohol.       Label    the 


Fig. 


-Simple  Water-Bath. 


This  is  a  useful  bath  for  individual 
workers.  It  is  provided  with  imbedding- 
cups,  infiltration  vials,  a  shelf  for  watch- 
glass  imbedding  or  for  warming  instru- 
ments, and  tubulatures  for  gas  regulator 
and  thermometer. 


ing  it   with  its  own  volume 

bottles  thick  and  thin  celloidin,  respectively. 

23.  Paraffin. — In  one  of  the  cups  of  a  warm  paraffin  oven 
(Fig.  24,  25,  or  26),  put  75  grams  of  paraffin,  melting  at  about 
53°  C.  The  bath  should  be  kept 
at  a  temperature  of  some  two 
degrees  above  the  melting-point 
of  the  paraffin.  A  supply  of  softer 
and  of  harder  paraffin  (e.  g.,  melt- 
ing at  43°  and  60°  C.)  should  also 
be  at  hand. 

Other  Reagents. — Provide  your- 
self with  200  c.c.  of  xylol,  25  c.c. 
of  clove  oil,  25  c.c.  of  glacial  acetic 
acid,  50  c.c.  of  cedar-wood  oil,  75 
c.c.  of  chloroform,  30  c.c.  of  gly- 
cerin   and    250    c.c.    of    absolute 


Imbedding-Table. 


There  should  be  two  rectangular 
boxes  (about  3X3X16  cm.)  to  contain 
paraffin.  When  in  use  the  boxes  are  so 
placed  on  the  imbedding-table  that  the 
paraffin  in  one  end  remains  melted ;  in 
the  other,  solid.  Regulate  the  tempera- 
ture by  placing  the  flame  at  the  proper 
distance  under  the  acute  angle  of  the 
table.  It  is  best,  when  gas  is  used,  al- 
ways to  turn  on  the  gas  completely  and 
then  regulate  the  height  of  the  flame  by 
means  of  a  clamp  on  the  rubber  tubing 
which  conducts  gas  to  the  burner. 


Chapter  I:   Preparation  of  Reagents  13 

alcohol  if  it  has  not  already  been  prepared.  Keep  the  absolute 
alcohol  and  the  xylol  carefully  corked  to  exclude  moisture. 
Before  measuring  out  any  of  these  reagents,  see  that  both  the 
graduate  and  bottle  are  perfectly  clean  and  dry. 

MEMORANDA 

1.  Ethyl  Alcohol  is  the  kind  commonly  used  in  histological  labora- 
tories. Upon  presentation  of  the  proper  credentials  to  the  internal 
revenue  officers,  it  may  be  purchased  by  the  barrel  from  distillers,  tax 
free,  by  educational  institutions.  Such  commercial  alcohol  is  of  about 
96  per  cent,  strength.  When  the  strength  is  unknown  it  should  be  tested 
by  means  of  an  alcoholometer  (see  2,  below). 

Methyl  Alcohol  (called  also  wood  alcohol  or  wood  spirits)  is  cheaper 
than  ethyl  alcohol  in  case  the  latter  cannot;  be  had  tax  free,  and  is  fairly 
satisfactory  in  most  cases.  It  is  poisonous  and  must  be  carefully  hand- 
led.    It  is  of  about  90  per  cent,  strength. 

Synthol  is  a  manufactured  product  now  on  the  market  which  seems 
to  answer  the  purposes  of  ordinary  absolute  alcohol.  It  is  designated  as 
a  synthetic  alcohol  by  its  manufacturers  and  is  cheaper  than  absolute 
alcohol. 

Rectified  Spirit  is  a  91  per  cent,  alcohol  (84  per  cent,  in  England). 

2.  The  Alcoholometer  is  a  convenient  instrument  for  determining  the 
strength  of  alcohol,  or  the  percentage  of  absolute  alcohol  in  a  spirituous 
mixture.  It  is  a  kind  of  hydrometer  with  a  scale  marked  to  indicate  the 
percentages  of  alcohol.  Different  strengths  of  alcohol  have  different 
specific  gravities,  consequently,  the  instrument  will  float  higher  or  lower 
in  the  liquid  depending  upon  the  percentage  of  alcohol  present.  The 
number  on  the  scale  just  at  the  surface  of  the  liquid  indicates  its  strength. 

3.  Rule  for  Dilution  of  a  given  strength  of  a  solution  with  a  lower 
per  cent,  of  the  same  solution.  (For  where  the  diluent  is  water,  i.  e., 
zero  per  cent.,  see  rule  under  reagent  1.)  Subtract  the  per  cent,  required 
from  the  per  cent,  of  the  solution  to  be  diluted;  also  subtract  the  per 
cent,  of  the  diluent  from  that  of  the  strength  required.  The  differences 
are  the  relative  proportions  of  the  diluent  and  the  solution  to  be  diluted 
that  must  be  used.  Thus,  to  prepare  a  35  per  cent,  solution  from  95  and 
20  per  cent,  solutions:  95-35  =  60;  35-20  =  15;  hence,  60  to  15,  or  4  to 
1  are  the  proportions  desired.  That  is,  4  parts  of  the  20  per  cent,  and  1 
part  of  the  95  per  cent,  solution  must  be  used  to  obtain  a  35  per  cent, 
solution. 

4.  "  To  Remove  Fixed  Stoppers,  take  the  bottle  in  the  left  hand  with  the 
forefinger  applied  to  one  side  of  the  stopper,  then  tap  the  other  side  of 


14  Animal  Micrology 

the  stopper  with  some  heavy  instrument,  such  as  the  handle  of  a  pocket- 
knife,  pressing  the  forefinger  against  the  direction  of  the  tap.  Turn  the 
bottle  round,  gradually  tapping  until  the  stopper  loosens.  Should  this 
device  prove  of  no  avail  (which  is  very  rarely),  hold  the  neck  of  the 
bottle  in  a  spirit  flame,  and  quickly  withdraw  the  stopper  as  the  glass  of 
the  neck  expands.  This  is  a  somewhat  risky  procedure,  but  is  very 
effectual  if  done  smartly."  {Journal  of  Applied  Microscopy,  Vol.  VI, 
p.  2116.)  The  glass  of  the  neck  may  be  more  safely  heated  by  looping 
a  heavy  cord  about  it  and  sawing  the  cord  back  and  forth  until  the  fric- 
tion warms  the  glass. 


CHAPTER  II 
GENERAL  STATEMENT  OF  METHODS 

Each  of  the  reagents  which  has  been  prepared  is  used  for  one 
or  more  of  the  purposes  to  be  discussed  in  this  chapter. 

All  methods  of  preparation  in  microscopy  are  to  enable  us  to 
learn  more  of  the  structure  and  functions  of  objects  than  would 
otherwise  be  apparent.  We  endeavor  to  study  them  in  as  near 
their  natural  condition  as  possible.  While  the  study  of  living  or 
of  fresh  material  is  desirable  it  can  be  carried  on  only  to  a  very 
limited  extent.  Most  structures  of  the  animal  body,  though 
opaque,  must  be  examined  largely  by  transmitted  light,  hence, 
special  preparation  is  necessary  to  put  them  into  suitable  condition. 
This  is  accomplished — 

1.  By  cutting  them  into  thin  slices  [section  method). 

2.  By  separating  them  into  their  elements   [isolation)  — 

a)  Mechanically  [teasing),  or 

b)  With  the  aid  of  fluids  which  remove  the  cement  sub- 
stance [dissociation  or  maceration). 

In  most  instances,  the  minute  structure  of  a  tissue  or  of  an 
organism  can  be  studied  to  the  best  advantage  only  after  the  appli- 
cation of  certain  agents  which  serve  to  emphasize  the  various  struc- 
tural elements.  A  tissue  so  prepared  is  an  artificial  product  in 
that  it  is  not  exactly  the  same  as  it  was  in  the  living  organism,  but 
recent  studies  of  protoplasm  in  the  living  condition  by  competent 
investigators  strengthen  the  belief  that  many  reagents  preserve 
very  faithfully  the  actual  structure  of  the  cell  contents.  The 
liquid  albuminoids  are  apparently  the  materials  which  suffer  the 
greatest  modifications.  Since  alterations  do  occur,  however,  it  is 
clear  that  in  our  interpretations  of  prepared  material  we  must 
reckon  carefully  with  both  the  original  nature  of  the  object  and 
with  the  factors  introduced  by  ourselves. 

15 


16  Animal  Micrology 

KILLING,  FIXING,  AND  HARDENING 
The  first  step  in  the  preparation  of  tissues  ordinarily  is  the 
employment  of  some  reagent  which  will  kill  the  tissues  and  fix 
their  various  components  in  the  characteristic  stages  of  their 
activities.  Such  material  may  then  be  preserved  indefinitely  for 
future  use. 

It  is  customary  to  discriminate  between  killing,  fixing,  and 
hardening,  although  the  same  reagent  may  fulfil  all  three  require- 
ments. Killing  refers  particularly  to  the  destruction  of  the  life 
of  the  tissue,  a  process  which  may  be  either  slow  or  instantaneous. 
In  slow  killing  it  is  usual  to  employ  narcotics  such  as  ether, 
chloroform,  chloral  hydrate,  chloretone,  carbon  dioxide,  nicotin, 
cocain,  or  weak  alcohol.  Ice  is  also  used  sometimes.  Such 
methods  are  of  particular  value  with  highly  contractile  animals 
which  are  desired  in  the  extended  condition.  Such  forms  are 
narcotized  completely  or  until  they  are  unable  to  contract  and 
then  frequently  fixed  and  hardened  in  other  or  stronger  fluids. 
Where  practicable,  instantaneous  killing  and  fixing  is  preferable 
because  tissues  have  then  no  time  to  undergo  postmortem  changes. 
The  same  fluid  ordinarily  is  employed  for  killing  and  fixing. 
The  purpose  of  fixation  is — 

a)  To  preserve  the  actual  form  of  tissue  elements. 

b)  To  produce  optical  differences  in  structure,  or  so  to  affect 
the  tissues  that  such  differences  will  be  brought  out  through  sub- 
sequent treatment  with  stains  or  other  reagents. 

To  accomplish  this  the  fixing  agent  must  possess  the  following 
qualities: 

1.  It  should  kill  the  tissue  so  quickly  that  few  structural 
changes  can  occur. 

2.  It  should  neither  shrink  nor  distend  the  tissue. 

3.  It  should  be  a  good  preservative ;  that  is,  it  must  render  the 
tissue  elements  insoluble  and  prevent  postmortem  changes. 

4.  It  should  penetrate  all  parts  equally  well. 

5.  It  should  put  the  tissue  in  condition  to  take  stains  unless  it 
of  itself  produces  sufficient  optical  differences  in  the  various  parts 
of  the  tissue. 


Chapter  II:    General  Statement  of  Methods  17 

No  ideal  single  reagent  has  been  discovered  which  meets  all 
of  these  requirements,  hence  it  is  customary  to  combine  two  or 
more  reagents  which  individually  possess  certain  of  these  desirable 
qualities.  All  of  the  best  fixing  reagents  are  mixtures.  For 
example,  acetic  acid  is  very  generally  used  in  fixing  mixtures 
because  it  penetrates  well,  produces  good  optical  differentiation, 
and  counteracts  the  tendency  of  some  reagents  (e.  g.,  corrosive 
sublimate)  to  shrink  tissues.  Again,  osmic  acid,  which  is  an  excel- 
lent fixing  agent  for  very  small  pieces  of  tissue,  penetrates  very 
poorly;  consequently  for  most  objects  it  must  be  mixed  with 
reagents  which  penetrate  rapidly  and  thoroughly. 

Some  fixing  agents  (corrosive  sublimate,  chromic  acid,  osmic 
acid,  etc.)  enter  into  chemical  combination  with  certain  of  the 
tissue  elements,  others  (alcohol,  picric  acid,  nitric  acid,  hot  water, 
etc.)  act  by  coagulating  or  precipitating  certain  constituents  of 
tissues. 

The  chief  object  of  hardening  is  to  bring  tissues  to  the  proper 
consistency  for  cutting  sections.  The  process,  although  begun 
ordinarily  by  the  fixing  agent,  is  usually  completed  in  alcohol. 
Some  objects  are  not  sufficiently  hardened  until  they  have  remained 
in  alcohol  for  many  hours,  or  even  days.  As  a  rule,  tissues  should 
remain  in  alcohol  of  at  least  70  per  cent,  strength  for  a  minimum 
of  24  hours  after  the  preliminary  operations  of  fixing,  washing,  etc., 
before  they  are  subjected  to  further  treatment. 

WASHING 

Fixing  agents  ordinarily,  with  the  exception  of  alcohol,  must 
be  washed  out  thoroughly  or  they  are  likely  to  interfere*  with  sub- 
sequent processes.  Aqueous  solutions  are  washed  out  usually  in 
water  or  a  low  per  cent,  of  alcohol;  alcoholic  solutions,  with  alco- 
hol of  about  the  same  strength  as  that  of  the  fixing  agent.  Wash- 
ing usually  requires  from  10  to  24  hours,  with  several  changes  of 
the  liquid.  If  water  is  the  washing  agent  it  is  best  where  prac- 
ticable to  use  running  water. 

Chromic  acid  and  its  compounds  should  be  washed  out  in  run- 
ning water.  This  should  be  done  in  the  dark  in  order  that  pre- 
cipitation may  be  avoided. 


18  Animal  Micrology 

Picric  acid,  or  solutions  containing  it,  must  be  washed  in 
strong  alcohol  (70  per  cent.),  never  in  water  because  the  latter 
seems  to  undo  the  work  of  fixation. 

Corrosive  sublimate  and  mixtures  containing  it  are  washed  out 
in  water  or  alcohol.  A  little  tincture  of  iodine  should  be  added 
to  the  wash  from  time  to  time  to  insure  the  removal  of  all  corro- 
sive sublimate  crystals.  Sufficient  iodine  has  been  added  when  it 
no  longer  loses  its  reddish  color  after  being  in  contact  with  the 
preparation  for  a  short  time. 

Osmic  acid  and  mixtures  containing  it  should  be  washed  in 

running  water. 

0 
DEHYDRATING 

While  under  certain  circumstances  objects  may  be  mounted  in 
aqueous  media  for  examination,  in  the  majority  of  cases,  especially 
where  the  preparation  is  to  be  a  permanent  one,  it  has  been  found 
best  to  remove  all  water  from  the  tissues,  that  is,  to  dehydrate 
them.  This  renders  preservation  more  certain,  and  it  is  a  neces- 
sity, moreover,  if  the  object  is  to  be  imbedded  later  in  paraffin  or 
celloidin,  for  neither  of  these  substances  is  miscible  with  water. 
Because  of  its  strong  affinity  for  water  and  the  ease  with  which  it 
may  be  manipulated,  alcohol  has  come  to  be  used  universally  for 
this  purpose.  It  completes  the  process  of  hardening  at  the  same 
time.  The  dehydration  must  be  gradual.  In  tissues  transferred 
from  water  or  aqueous  solutions  directly  to  strong  alcohol  (or  vice 
versa)  violent  diffusion  currents  are  set  up  which  produce  serious 
distortion  of  the  tissue  elements.  For  this  reason  a  series  of 
alcohols  of  gradually  increasing  strength  (e.  g.,  35-50-70-83-95 
per  cent.)  is  used.  The  more  delicate  the  object,  the  closer  should 
be  the  grades  of  alcohol. 

PRESERVING 

After  fixing  and  washing,  the  process  of  dehydration  is  begun 
ordinarily  and  tissues  are  carried  as  far  as  70  per  cent,  alcohol. 
It  is  customary  to  leave  them  in  alcohol  of  from  70  to  83  per 
cent,  strength  until  they  are  needed.  They  may  remain  here 
indefinitely.     If  they  are  to  be  preserved  for  a  long   time  (for 


Cuapter  II:    General  Statement  of  Methods  19 

months) ,  however,  it  is  better  to  keep  them  in  a  mixture  of  equal 
parts  of  glycerin,  distilled  water,  and  strong  (commercial) 
alcohol. 

STAINING 

A  few  fixing  agents  produce  sufficient  optical  differentiation  in 
tissues,  but  as  a  rule  this  must  be  accomplished  through  the  addi- 
tion of  certain  stains.  Most  of  the  stains  used  have  more  or  less 
of  a  selective  action;  that  is,  they  pick  out  certain  elements  of  the 
tissue,  and  thus  enable  one  to  see  details  of  structure  that  would 
otherwise  be  invisible.  Their  action,  however,  depends  largely 
upon  the  nature  of  the  fixing  agent  which  has  previously  been 
used.  The  secret  of  good  staining,  indeed,  lies  largely  in  proper 
fixation. 

There  are  large  numbers  of  stains  of  very  different  chemical 
constitution  (acid,  neutral,  and  alkali),  and  they  may  act  in  very 
different  ways  upon  the  material  to  be  stained.  For  example, 
some  show  affinity  only  for  certain  elements  of  the  nucleus,  others 
for  the  cytoplasm  of  cells,  and  some  are  present  in  tissues  only 
physically  as  deposits,  while  others  enter  into  chemical  combina 
tion  with  certain  of  the  cell  constituents.  A  few,  such  as  borax- 
carmine,  are  general  stains,  and  affect  to  a  greater  or  less  degree 
practically  all  the  tissue  elements. 

It  is  not  the  purpose  of  the  present  book  to  enter  into  a  pro- 
longed discussion  of  the  theory  of  staining  or  to  undertake  a 
description  and  classification  of  stains.     For  this  the  reader  is 
referred  to  the  excellent  compendium  of  Lee  (The  Microtomisfs 
.    Vade-Mecum). 

The  stains  of  widest  application  are  (1)  the  Carmines,  (2)  the 
\  Hematoxylins,  (3)  the  Anilins,  and  (4)  Metallic  substances. 

Carmine  is  a  brilliant  scarlet  or  purplish  coloring  matter  made 
from  the  bodies  of  the  cochineal  and  kermes  scale  insects.  The 
carmine  stains,  including  cochineal,  have  been  largely  used  in  the 
past  for  all  kinds  of  work,  but  at  present  they  are  used  more  par- 
ticularly for  staining  objects  in  bulk  before  sectioning,  or  objects 
which  are  not  to  be  sectioned.  They  are  easy  to  use,  and  will 
follow  almost  any  fixing  agent.     In  case  of  over-staining,  weak 


20  Animal  Micrology 

hydrochloric  acid  (0.1  to  1  per  cent.)  is  used  to  decolorize  the 
tissues.     For  formulae  see  Appendix  B. 

Hematoxylin  is  a  compound  containing  the  coloring  matter  of 
logwood.  The  hematoxylins  follow  well  almost  any  of  the  fixing 
agents;  they  are  especially  recommended  after  fluids  containing 
chromic  acid  or  its  salts.  According  to  Mayer,  the  active  agent 
in  these  stains  is  a  compound  of  hematein  with  alumina.  The 
hematein  is  produced  by  the  oxidation  of  hematoxylin.  The  so- 
called  "ripening"  is  simply  this  change,  which  is  brought  about 
by  exposing  the  hematoxylin  solution  to  air.  If  the  pure  hematein 
is  used  in  making  the  stain,  therefore,  the  latter  will  be  ready 
for  use  immediately,  because  it  need  not  undergo  the  ripening 
process  (see  reagent  47,  Appendix  B).  For  formulae  see 
Appendix  B. 

Anilin  is  a  colorless  coal-tar  derivative,  and  is  the  base  from 
which  many  of  the  numerous  coal-tar  dyes  are  made.  The  anilins 
are  brilliant  stains  of  all  colors.  They  are  used  almost  exclusively 
for  staining  sections  or  thin  membranes,  and  are  of  great  service 
to  the  microscopist,  although,  as  a  rule,  they  fade  in  time. 

The  basic  anilin  stains,  such  as  methyl  green,  methyl  violet, 
gentian  violet,  methylen  blue,  safranin,  Bismarck  brown,  toluidin 
blue,  and  thionin  are  usually  nuclear  stains.  On  the  other  hand, 
the  acid  anilin  stains,  such  as  acid  fuchsin,  eosin,  erythrosin,  light 
green,  orange  G,  bleu  de  Lyon,  nigrosin,  benzopurpurin,  and 
aurantia  are  ranked  as  cytoplasmic  stains.  These  stains  must  be 
made  up  fresh  every  two  or  three  weeks,  as  they  frequently  spoil 
if  kept  much  longer. 

The  metallic  substances  used  for  color  differentiation  operate 
principally  as  impregnations  rather  than  as  stains.  The  coloring 
matter  is  held  physically  as  a  precipitate  or  reduction  product  in 
certain  of  the  tissue  elements.  The  commonest  reagents  of  this 
class  in  use  are  silver  nitrate  and  gold  chloride. 

The  different  tissue  elements  frequently  show  affinity  for 
different  stains,  consequently  it  is  a  common  practice  to  use  more 
than  one  stain.  Very  decided  contrasts  may  thus  be  produced, 
such  as  red  and  blue,  red  and  green,  green  and  orange,  etc.     It 


Chapter  II:   General  Statement  of  Methods  21 

is  not  uncommon,  in  fact,  to  have  triple  and  even  multiple  staining. 
In  such  staining,  the  stains  are  sometimes  applied  consecutively,; 
in  other  cases,  at  different  points  in  the  process  of  general  manipu- 
lation. Sometimes  all  the  stains  may  be  mixed  together,  so  that 
immersion  of  the  sections  in  one  liquid  is  all  that  is  required  for 
double  or  multiple  staining. 

A  general  rule  in  staining,  especially  for  entire  or  bulky 
objects,  is  that  the  specimen  should  be  transferred  to  the  stain 
from  a  reagent  in  which  the  percentage  of  water  is  approximately 
the  same  as  that  of  the  stain.  The  same  is  true  when  the 
object  is  removed  from  the  stain.  For  example,  if  the  stain  to  be 
used  is  an  aqueous  solution,  the  object  should  enter  it  from  an 
aqueous  solution;  if  the  stain  is  made  up  in  95  per  cent,  alcohol, 
the  object  should  enter  from  95  per  cent,  alcohol,  etc.  For 
reasons  see  "dehydrating." 

CLEARING 

In  the  vast  majority  of  cases  tissues  are  too  opaque  for  satis- 
factory examination  until  they  have  been  treated  with  certain 
clarifying  reagents  or  clearers  which  render  them  more  trans- 
parent. 

Such  reagents  as  glycerin,  glycerin -jelly,  etc., '  are  used 
when  the  object  is  to  be  cleared,  without  alcoholic  dehydration, 
directly  from  water.  Usually,  for  permanent  preparations,  the 
alcoholic  dehydration  method  is  employed  and  it  then  becomes 
necessary  to  use  a  clarifying  reagent  which  will  replace  the 
alcohol  and  facilitate  the  penetration  of  the  final  mounting-medium 
(balsam  or  damar). 

Perhaps  the  most  useful  and  rapid  clearer  is  xylol.  Xylol, 
however,  is  very  sensitive  to  moisture  and  if  the  preparation  has 
not  been  thoroughly  dehydrated  the  final  mount  will  appear 
milky.  For  this  reason  the  beginner  is  recommended  to  use  a 
carbol-xylol  mixture  (see  reagent  9,  chap.  i).  Carbolic  acid  has 
a  great  affinity  for  water,  and  the  mixture  will  therefore  clear 
preparations  that  are  not  fully  dehydrated.  Cedar-wood  oil, 
though  somewhat  slower  than  xylol,  is  one  of  the  best  clearers. 
It  is  also  one  of  the  safest,  because  tissues  may  be  left  in  it 


22  Animal  Micrology 

indefinitely.  Other  good  clearers  after  alcohol  are  oil  of  origanum, 
sandal-wood  oil,  oil  of  cloves,  toluol,  oil  of  bergamot,  anilin  oil 
(for  watery  specimens),  carbolic  acid  (for  watery  specimens),  and 
beech  wood  creasote.  Clove  oil  should  not  be  used  for  celloidin 
sections  because  it  dissolves  celloidin.  It  is  also  inapplicable 
ordinarily  after  most  anilin  dyes  because  of  its  tendency  to 
extract  them.  Among  the  best  reagents  for  celloidin  sections 
are  cedar- wood  oil,  carbol-xylol,  oil  of  origanum,  creasote,  and 
Eycleshymer's  clearer  (memorandum  4,  chap.  vii). 

While  "clearing"  refers  especially  to  the  rendering  trans- 
parent of  tissue  elements,  and  dealcoholization  to  the  removal  of 
alcohol  previous  to  imbedding  in  paraffin,  very  frequently  the 
same  reagent  is  used  for  either  purpose  and  the  term  " clearing" 
has  come  to  be  used  in  either  sense. 

MOUNTING 

After  tissues  have  been  cleared  the  final  step  is  to  mount  them 
in  some  suitable  medium  for  preservation  and  inspection. 

If  tissues  are  to  be  mounted  directly  from  water  or  aqueous 
media,  glycerin,  glycerin-jelly,  or  Farrant's  solution  is  used  ordi- 
narily. If  the  alcoholic  dehydration  method  is  employed,  balsam 
or  gum  damar  is  the  final  mounting  medium.  The  balsam  or 
damar  is  dissolved  commonly  in  xylol,  although  turpentine,  chlo- 
roform, or  benzol  may  be  used  as  the  solvent.  Xylol -balsam  is 
the  most  satisfactory  for  ordinary  purposes. 

IMBEDDING 

In  order  to  section  tissues  or  objects  satisfactorily  it  is  fre- 
quently necessary  to  imbed  them  in  a  suitable  matrix.  Simple 
imbedding  consists  in  merely  surrounding  the  object  by  an 
appropriate  medium  to  hold  it  in  place  while  it  is  being  cut.  In 
interstitial  imbedding  the  object  is  saturated  {infiltrated)  with 
the  imbedding  substance  which,  when  all  cavities  and  inter- 
sticies  are  filled,  is  caused  to  set;  thus  it  supports  all  parts  of 
the  tissue  and  holds  the  components  in  place  when  sections  are 
made.  Infiltration  imbedding  is  of  great  importance  to  micros- 
copists  and  much  of  the  space  of  the  present  book  is  given  up  to 


Chapter  II:   General  Statement  of  Methods  23 

drilling  the  student  in  the  details  of  the  two  chief  infiltration 
methods,  viz.,  the  paraffin  method  and  the  celloidin  method. 
Infiltration  with  gum  is  also  not  infrequently  resorted  to,  espe- 
cially for  tissues  which  would  be  injured  by  alcohol,  or  for 
sectioning  by  the  freezing  method. 

Paraffin  is  a  translucent,  waxy  material  derived  from  various 
sources,  one  of  the  commonest  of  which  is  crude  petroleum. 
Paraffins  of  low  and  of  high  melting-points,  termed  respectively 
soft  and  hard  paraffin,  should  be  kept  on  hand  so  that  mixtures 
of  different  degrees  of  hardness  may  be  made  up  as  necessity 
demands. 

Celloidin  is  a  form  of  pyroxilin  (gun  cotton  or  collodion 
cotton)  specially  prepared  for  interstitial  imbedding.  It  is  dis- 
solved in  a  mixture  of  ether  and  alcohol  (chap,  i,  reagent  4)  and 
solutions  of  two  or  three  strengths  are  used  for  infiltration.  For 
details  see  the  method,  chap.  vii.  Collodion  instead  of  celloidin 
is  used  by  some  workers  (see  memorandum  11,  chap.  vii). 

AFFIXING  SECTIONS 

When  mounting  sections  upon  a  slide,  especially  if  they  are 
yet  to  be  stained,  it  is  usually  necessary  to  affix  them  firmly  to 
the  slide  to  prevent  later  displacement.  For  paraffin  sections 
Mayer's  albumen  fixative  (reagent  21,  chap,  i),  or  a  combination 
of  this  method  with  the  water  method,  is  most  widely  used.  The 
water  method  alone  often  proves  adequate,  particularly  with  thin 
sections.  The  slide  is  flooded  with  water  and  the  sections  are 
floated  upon  its  surface.  As  the  layer  of  water  evaporates  the 
sections  are  slowly  drawn  down  into  close  contact  with  the  slide. 
When  perfectly  dry  they  are  usually  so  firmly  affixed  that  they  will 
not  become  detached  even  after  the  removal  of  paraffin  from  them. 
It  is  common,  however,  and  safer  to  use  a  thin  film  of  albumen 
fixative  as  a  cementing  substance  between  the  water  and  the 
surface  of  the  slide. 

In  the  case  of  celloidin  sections,  if  only  one  or  a  few  sections 
are  to  be  mounted  on  one  slide,  it  is  a  common  practice  to  stain 
the  sections  and  transfer  them  through  the  various  reagents,  even 


24  Animal  Micrology 

to  clearing,  before  mounting  them  on  the  slide.  In  such  cases 
the  sections  need  not  be  fixed  to  the  slide.  With  serial  sections, 
however,  the  sections  must  be  held  in  place  some  way  during 
their  transition  through  the  reagents  (see  memorandum  12, 
chap,  vii) .  Unlike  paraffin,  the  celloidin  is  not  ordinarily  removed 
from  the  tissues. 

DECOLORIZING 

Not  infrequently  in  staining  the  tissue  becomes  overstained 
and  requires  that  some  of  the  color  be  extracted  from  certain  of 
the  elements  to  bring  about  a  proper  differentiation.  The  fact 
that  certain  tissue  elements  retain  stain  more  tenaciously  than 
others  is  sometimes  taken  advantage  of  and  overstaining  followed 
by  decolorization  is  practiced  intentionally.  Alcohol  slightly 
acidulated  with  hydrochloric  acid  (0.1  to  1  per  cent.)  is  commonly 
used  for  the  extraction  of  surplus  color.  In  special  cases  other 
decolorizers  are  used:  for  example,  iron-alum  in  the  iron-hema- 
toxylin  method  (reagent  18,  chap.  i). 

BLEACHING 

In  some  cases,  tissues  are  obscured  because  of  the  presence  of 
natural  pigments  or  on  account  of  blackening  caused  by  the  fixing 
reagent.  Such  tissues  must  be  bleached.  Chlorine,  peroxide  of 
hydrogen,  or  sulphurous  acid  are  commonly  employed.  A  method 
is  given  in  chap,  v,  memorandum  12. 

CORROSION 

To  obtain  skeletal  structures,  as  for  example  the  spicules  of 
sponges  or  the  hard  parts  of  insects,  various  methods  of  corrosion 
are  employed.  Nitric  acid,  caustic  potash,  caustic  soda,  eau  de 
Javelle  are  reagents  often  used  for  this  purpose.  Corrosive  prep- 
arations of  injected  vessels  and  cavities  may  also  be  made. 
DECALCIFICATION  AND  DESILICIDATION 

Tissues  impregnated  with  lime  salts  or  with  silica  must  have 
such  hard  parts  removed  usually  before  they  can  be  sectioned. 
For  decalcification,  one  of  several  acids  may  be  used.  The  details 
are  given  in  the  chapter  on  bone,  tooth,  etc.  (chap.  xi).  For  de- 
calcifying reagents,  see  Appendix  B,  v. 


Chapter  II:   General  Statement  of  Methods  25 

Where  desilicidation  is  necessary  hydrofluoric  acid  may  be  em- 
ployed, although,  because  of  its  property  of  attacking  mucous 
membranes,  its  use  is  attended  with  more  or  less  danger  for  the 
operator.  It  is  added  drop  by  drop  to  the  tissue  which  has  pre- 
viously been  placed  in  a  paraffin-coated  vessel  (the  acid  attacks 
glass).  If  the  tissue  is  not  too  heavily  impregnated  with  silica, 
it  is  safer  to  use  an  old  section  razor  and  try  to  cut  sections  with- 
out previously  treating  them  with  hydrofluoric  acid. 

INJECTION  METHODS 

The  injection  of  colored  masses  into  the  blood  vessels  and 
other  vessels  of  the  body  is  frequently  practiced  to  aid  in  deter- 
mining their  distribution  and  their  relation  to  the  surrounding 
tissues.  The  dye  is  termed  the  coloring  mass  and  the  substance 
to  which  it  is  added,  the  vehicle. 

ISOLATION  OF  HISTOLOGICAL  ELEMENTS 

Isolation  is  one  of  the  most  valuable  means  of  forming  a  cor- 
rect conception  of  cells  and  fibers.  It  has  the  advantage  over 
sections  that  the  elements  may  be  inspected  in  their  entirety  and 
from  all  sides.  The  separation  is  accomplished,  as  already  noted, 
by  (1)  reagents  which  dissolve  or  soften  cell  cement  and  inter- 
stitial material  without  seriously  affecting  the  cells  (maceration 
or  dissociation),  or  (2)  mechanically  by  means  of  dissecting  needles 
(teasing),  or  both.  Hardening  and  fixing  reagents  in  general  if 
diluted  to  about  one-tenth  are  efficient  for  dissociation.  Gage 
recommends  normal  saline  as  preferable  to  water  for  dilution. 
The  dissecting  microscope  or  some  kind  of  lens-holder  and  lens 
are  valuable  aids  in  isolating  tissue  elements.  For  practical 
methods  consult  chap,  x ;  for  reagents,  Appendix  B,  iv. 

NORMAL  OR  INDIFFERENT  FLUIDS  FOR  EXAMINING  FRESH  TISSUES 

It  is  desirable  frequently  to  examine  fresh  material  in  as  near 
a  natural  condition  as  possible,  hence  recourse  is  had  to  the  so- 
called  indifferent  fluids.  While  not  wholly  indifferent,  they  ordi- 
narily produce  but  slight  changes  in  tissues  and  their  elements 
from  the  view-point  of  the  microscopist.  The  liquids  most  com- 
monly used  for  this  purpose  are  discussed  in  Appendix  B,  iii. 


26 


Animal  Micrology 


GENERAL  SCHEME  FOR  MOUNTING  WHOLE  OBJECTS  (IN  TOTO  PREP- 
ARATIONS) OR  SECTIONS 

Whole  Objects  (for  balsam  mounts)    Section  Methods  (paraffin  and  celloidin) 

Killing  and  fixing  Killing  and  fixing 

I  I 

Washing  Washing 

Staining  (Staining,  if  to  be  stained  in  bulk) 

(Decolorizing  if  necessary)  Hardening  and  dehydrating 

I  I 

Dehydrating  Absolute  alcohol 


Clearing 
Mounting 


If  not  stained 
in  bulk 

Through  alco- 
hols to  stain 
I 

Staining 

Washing 

Dehydrating 
( and    decol- 
o  r  i  z  i  n  g  if 
necessary) 


Paraffin  Method 
Dealcoholization  (xylol) 

Melted  paraffin 

Imbedding 

Sectioning 

I   . 
Affixing  sections 

I 
Removal  of  paraffin 

I 
Absolute  alcohol 

Clearing 

I 
Mounting 


If  not  stained  in 

bulk 
Staining 

I 
Washing  (and  de- 
colorizing  if 
necessary) 


Celloidin  Method 
Ether-alcohol 

Thin  celloidin 

Thick  celloidin 

I 
Imbedding 

Sectioning  * 


Dehydrating 
95  per  cent, 
cohol 

I 
Clearing 

Mounting 


to 


*  If  sections  are  to  be  arranged  serially  they  must  be  affixed  to  the  slide  as  soon  as  cut. 


CHAPTER  III 

KILLING  AND  FIXING 

Cautions. — 1.  Use  only  fresh  tissues  and  work  rapidly  so 
that  the  tissue  elements  will  not  have  time  to  undergo  postmortem 
changes. 

2.  Remove  organs  carefully,  and  avoid  crushing  or  pressing 
the  parts  to  be  prepared. 

3.  Tissues  should  never  be  allowed  to  dry  from  the  time  they 
leave  the  animal  until  they  are  finally  mounted  for  microscopical 
examination  except  at  one  point  in  the  paraffin  method. 

4.  Use  only  small  pieces  (2  to  6  mm.  cube)  of  tissue  whenever 
possible,  or  penetration  of  the  reagent  will  be  insufficient. 
Embryos  and  small  objects  up  to  4  cm.  in  size  may  be  placed 
entire  in  certain  oj  the  fixing  fluids. 

5.  For  fixing  and  hardening,  the  bulk  of  the  fluid  should  be 
from  10  to  50  times  that  of  the  object.  Too  many  pieces  should 
not  be  placed  in  the  same  vial. 

6.  Use  only  clean  reagents.  It  is  well  to  let  the  object  rest  on 
a  bit  of  cotton  in  the  bottom  of  the  vial  or  have  it  suspended  from 
the  vial  mouth  so  that  the  reagent  may  penetrate  equally  from  all 
sides.     Penetration  is  aided  by  heat. 

7.  When  necessary  to  wash  fresh  tissue,  it  is  usually  best  to 
use  normal  saline,  and  not  water.  Let  it  flow  gently  over  the 
surface  of  the  object  or  slowly  twirl  the  latter  in  the  fluid.  Do 
not  scrape  off  foreign  matter. 

8.  In  many  cases  the  killing  and  fixing  reagent  does  not 
harden  the  tissue  sufficiently  and  the  hardening  process  must  be 
completed  in  alcohol. 

9.  Keep  the  reagents  and  preparations  from  direct  sunlight. 

10.  Carefully  label  each  vessel  containing  tissue.  State  the 
contents,  the  fluid  used,  and  the  date.     Label  on  the  side. 

11.  Keep  a  careful  record  on  cards  of  the  reagents  used,  and 
the  time  tuhen  changed,  for  each  separate  piece  of  tissue. 

.      27 


28  Animal  Micrology 

PRACTICAL   EXERCISE 

Kill  a  frog  by  placing  it  under  a  bell  jar  which  contains  a  bit 
of  cotton  saturated  with  chloroform.  Open  the  body  as  soon  as 
possible  after  death  and  secure  the  tissues  specified  below. 

1.  Alcohol  Fixation. — Remove  the  dorsal  aorta  and  small 
pieces  of  the  liver  and  harden  in  absolute  alcohol  (at  least,  not 
less  than  95  per  cent.)  in  a  vial  or  small  bottle.  The  tissue  will 
be  ready  for  further  treatment  in  two  days. 

Larger  pieces  of  tissue  require  longer  time.  The  pieces  should 
be  thin.     Change  the  alcohol  every  day  for  the  first  three  days. 

Alcohol  is  in  many  instances  an  unsatisfactory  fixing  reagent, 
but  it  is  frequently  employed  because  it  is  usually  at  hand  and  is 
easily  manipulated.  Hot  absolute  alcohol  is  very  often  used  for 
insects.  If  absolute  alcohol  is  used,  the  fixation  may  be  fairly 
good,  but  because  of  the  expense  attached  to  the  best  absolute 
alcohol,  the  lower  percentages  are  more  frequently  used.  They 
shrink  protoplasm,  however,  and  are  not  to  be  recommended  for 
the  finer  histological  work.  Ninety-five  per  cent,  alcohol  is  as 
low  as  should  be  used  for  fixing,  although  70  per  cent,  is  sufficient 
to  preserve  specimens  for  other  than  microscopical  work.  Acetic 
acid  (Appendix  B,  2)  is  used  with  alcohol  sometimes  to  increase 
penetration  and  to  counteract  its  tendency  to  shrink  tissues.  The 
mixture  is  usually  preferable  to  alcohol  alone. 

2.  Fixing  with  Gilson's  Mercuro-Nitric  Mixture. — Place  small 
pieces  of  liver,  kidney,  pancreas,  esophagus,  cardiac  and  pylo- 
ric ends  of  the  stomach,  apex  of  the  heart,  bladder,  testis  or 
ovary,  and  tongue  in  Gilson  for  from  two  to  six  hours.  Remove 
a  piece  of  intestine  about  12  mm.  long,  and  after  washing  it 
thoroughly  in  normal  saline  place  it  in  a  small  vial  containing 
about  fifty  times  its  bulk  of  fixing  mixture  and  leave  it  for  two 
hours.  After  fixation  wash  the  objects  thoroughly  in  water  fol- 
lowed by  35  and  50  per  cent,  alcohol  (15  minutes  each),  and  pre- 
serve them  in  70  per  cent,  alcohol.  Read  remarks  on  washing 
out  corrosive  sublimate,  Appendix  B,  reagent  13,  caution  1. 

Gilson's  is  an  excellent  general  reagent  and  gives  a  very  deli- 
cate fixation.     It  is  perhaps  the  most  satisfactory  killing  and  fix- 


Chapter  III:   Killing  and  Fixing  29 

ing  reagent  that  the  beginner  can  use.  The  time  which  objects 
should  be  left  in  the  fluid  varies  from  ten  or  fifteen  minutes^or 
very  delicate  objects  to  six  hours  for  larger  or  denser  tissues, 
although  many  objects  may  be  left  for  thirty-six  hours  without 
injury.  When  an  object  becomes  opaque  throughout  it  is  suffi- 
ciently fixed.  This  holds  true  of  other  corrosive  sublimate  fixing 
fluids.  Corrosive  sublimate  alone  is  also  widely  used  as  a  general 
reagent.     See  caution  2  under  reagent  13  in  Appendix  B. 

3.  Fixing  with  Erlicki's  Fluid. — Remove  a  small  piece  of  the 
spinal  cord  1  cm.  in  length  and  place  it  in  about  one  hundred 
times  its  volume  of  Erlicki's  fluid.  Likewise  place  the  brain  in 
this  fluid.  The  spinal  cord  must  remain  about  five  days  and  the 
brain  a  week  or  ten  days  in  the  liquid.  At  the  end  of  this  time 
transfer  the  object  to  35  per  cent,  alcohol,  keeping  it  in  the  dark 
for  two  hours  to  avoid  precipitation.  The  alcohol  should  be 
changed  occasionally  during  this  time.  Repeat  the  process  using 
50  per  cent,  alcohol,  and  finally  preserve  the  material  in  70  per 
cent,  alcohol. 

Erlicki's  fluid  is  an  excellent  reagent  for  general  use,  and  is 
especially  valuable  for  voluminous  objects  such  as  advanced 
embryos.  Its  principal  drawback  is  the  length  of  time  required 
properly  to  harden  objects  (ten  days  to  three  weeks  for  objects 
larger  than  the  above  tissues).  The  process  may  be  hastened  by 
keeping  the  fluid  containing  the  tissue  at  the  temperature  of  an 
incubator  (39°  C). 

4.  Formalin  as  a  Fixing  Reagent. — Place  a  piece  of  spinal 
cord,  liver,  and  fragments  of  muscle  in  which  nerves  terminate  in 
10  per  cent,  formalin  and  leave  until  needed  for  work  later. 
Formalin  in  varying  percentages  is  widely  used  for  the  preserva- 
tion and  fixation  of  specimens  for  dissection.  It  is  especially 
serviceable  for  the  central  nervous  system.  Most  specimens  may 
remain  in  it  indefinitely  without  injury.  For  simple  preservation, 
solutions  ranging  from  2  to  5  per  cent,  are  adequate,  but  for  fixa- 
tion, it  should  be  stronger  (10  per  cent.).  Entire  human  brains 
may  be  fixed  and  hardened  in  a  10  per  cent,  solution  with  fairly 
good  results. 


30  Animal  Micrology 

MEMORANDA 

1.  Tissues  Are  Preserved  in  Alcohol  of  from  70  to  85  per  cent,  strength, 
but  if  they  are  to  remain  several  months  it  is  better  to  preserve  them  in 
a  mixture  of  equal  parts  of  glycerin,  distilled  water,  and  95  per  cent, 
alcohol. 

2.  Hardening. — Read  carefully  the  remarks  on  hardening  in  chap.  ii. 

3.  Tissues  Should  Not  Be  Left  in  the  Fixing  Agent  longer,  ordinarily, 
than  is  necessary  to  get  results.  Some,  however,  require  a  long  time  to 
bring  out  the  optical  differences  of  their  elements.  Experience  alone 
can  teach  the  time  required  in  a  given  case.  Such  a  reagent  as  formalin 
kills,  fixes,  hardens,  and  preserves,  all  at  the  same  time. 

4.  For  Transferring  Small  Objects  through  reagents  the  method  of 
Walton  is  an  excellent  one.  For  the  several  reagents,  he  uses  shell 
vials  which  measure  about  10  cm.  in  height  by  3  cm.  in  diameter. 
Through  the  center  of  a  flat  cork  which  fits  the  vials,  a  hole  is  made  and 
a  glass  tube  (about  9  cm.  by  1.5  cm.)  is  inserted  so  that  its  lower  end  dips 
well  into  the  reagents  in  the  vials.  The  lower  end  of  the  tube  is  closed 
with  fine-meshed  cloth  and  the  objects  are  placed  within  the  tube.  To 
transfer  the  objects  one  simply  removes  the  cork  bearing  the  tube,  and 
inserts  it  in  the  vial  containing  the  desired  reagent.  The  upper  end  of 
the  tube  may  be  closed  with  a  cork  of  the  proper  size.  To  avoid  disturb- 
ance from  changes  in  air  pressure  a  small  hole  should  be  bored  in  the 
side  of  the  tube  just  below  the  lower  level  of  the  larger  cork.  The  vials 
are  supported  as  indicated  in  memorandum  5. 

5.  Shell  Vials,  Small  Bottles,  etc.,  when  in  use  are  best  supported 
in  shallow  auger  holes  of  proper  size  in  thick  blocks  of  wood. 

6.  Material  Which  Is  To  Be  Kept  Indefinitely  should  be  put  in  tightly 
stoppered  vials  in  a  place  away  from  strong  light.  It  is  best  to  pack  the 
vials  in  a  museum  jar  on  cotton  and  then  seal  the  jar  securely  to  prevent 
evaporation.  Material  is  even  more  secure  if  the  museum  jar  is  partly 
filled  with  alcohol;  in  such  a  case  each  small  vial  should  have  a  label  of 
the  contents  placed  within  it. 

Another  way  to  prevent  evaporation  from  vials  or  bottles  is  to  "  cap  " 
them  with  a  suitable  varnish  (see  7). 

7.  To  Seal  Bottles  and  Preparation  Jars  ("bottle-capping")  dip  the 
stopper  and  part  of  the  neck  in  collodion  varnish  made  as  follows: 

Pyroxylin 1  oz. 

Ether        6  oz. 

Alcohol 8  oz. 

When  the  pyroxylin  has  completely  dissolved  add  2.5  drams  of  camphor. 
(From  Pharmaceutical  Era,  Vol.  XXX,  p.  528.) 


Chapter  III:   Killing  and  Fixing  31 

8.  For  the  Preservation  of  Anatomical  Specimens  for  other  than  histo- 
logical purposes,  Gait  {The  Lancet,  Nov.  16, 1901,  p.  1334)  recommends 
the  following  fluid  as  superior  to  the  well-known  Kaiserling's  fluid. 

Sodium  chloride 5  parts 

Potassium  nitrate 1  part 

Chloral  hydrate       1  part 

Water 100  parts 

Wash  fresh  tissues  for  several  hours  in  running  water,  then  "  set "  in  an 
excess  of  methyl  alcohol  to  which  0.5  per  cent,  formalin  has  been  added 
(time  required:  six  hours  to  a  week  according  to  nature  and  size  of  spe- 
cimen). Next  transfer  the  specimen  directly  to  the  preserving  fluid, 
changing  the  latter  after  two  or  three  weeks  if  necessary.  In  case  the 
preparation  is  not  sealed,  sufficient  water  to  make  up  loss  by  evaporation 
must  be  added  occasionally.  Specimens  are  said  to  retain  their  natural 
colors. 

The  following  mixture,  recommended  to  the  author  by  Professor 
Kincaid  of  the  Washington  State  University,  has  given  most  excellent 
results.  To  a  mixture  of  equal  parts  of  glycerin  and  strong  alcohol 
sufficient  formalin  is  added  to  make  the  whole  about  a  2  per  cent,  forma- 
lin. Specimens  remain  perfectly  flexible  in  this  mixture,  and,  indeed, 
after  they  have  become  thoroughly  saturated,  many  forms  (crustacea, 
insects,  etc.)  may  be  removed  and  kept  as  dry  specimens  which  still 
retain  their  flexibility. 


CHAPTER  IV 
SIMPLE  SECTION  METHODS 

FREE  HAND  SECTION  CUTTING 
This  method  is  important  because  it  requires  no  costly  appli- 
ances; although  the  sections  are  not  as  accurately  cut  as  when 
mechanical    aids    are   used,    the    method   is    simple,    rapid,  and 
adequate  for  the  more  general  histological  and  pathological  work. 

1.  The  section  razor  is  flat  on  one  side  (the  lower),  and 
hollow  ground  on  the  other  (Fig.  15) .     It  must  be  sharp. 

2.  A  shallow  glass  dish  or  watch-glass  partly  filled  with 
water  is  also  necessary.  Before  making  a  section  dip  the  razor 
flatwise  into  the  liquid,  or  use  a  camel's  hair  brush;  see  that  the 
upper  surface  is  well  flooded. 

3.  Sit  in  such  a  way  that  the  fore-arm  may  be  steadied 
against  the  edge  of  the  table. 

4.  Use  a  piece  of  liver  which  was  fixed  in  formalin,  first 
rinsing  it  in  water.  Take  the  tissue  between  the  thumb  and 
forefinger  of  the  left  hand,  and  hold  it  in  such  a  way  that  a  thin 
slice  may  be  cut  by  drawing  the  knife  along  the  surface  of  the 
forefinger. 

5.  Rest  the  flat  surface  of  the  knife  upon  the  forefinger,  and, 
beginning  at  the  heel  of  the  knife,  carefully  draw  the  blade 
toward  you  diagonally  through  the  tissue,  slicing  off  a  thin 
section  of  as  uniform  thickness  as  possible. 

6.  As  each  section  is  cut,  float  it  off  into  the  water;  if  it  adheres 
to  the  blade,  remove  it  by  means  of  a  wet  camel's  hair  brush. 

7.  Practice  until  very  thin  sections  are  obtained,  then  place 
the  dish  upon  a  black  surface,  and  with  a  needle  or  section  lifter 
transfer  the  thinnest  and  best  sections,  if  only  fragments,  to  a 
watch-glass  containing  water. 

Note. — In  case  the  tissue  has  been  preserved  in  alco&ol,  cut  the  sec- 
tions under  70  per  cent,  alcohol  instead  of  water,  then  transfer  them  to 
50  and  35  per  cent,  alcohol  successively  and  finally  to  water,  leaving 
them  in  each  liquid  from  3  to  5  minutes. 

33 


34  Animal  Micrology 

8.  Next,  place  the  sections  in  about  3  c.c.  of  Delafield's  hema- 
toxylin diluted  with  an  equal  volume  of  water,  and  leave  them  for 
various  lengths  of  time  (3,  7,  12  minutes)  to  determine  the  time 
for  successful  staining. 

9.  Transfer  the  sections  from  the  stain  to  tap  water,  and  gently 
move  them  about  for  from  5  to  10  minutes  to  wash  out  the  excess 
of  the  stain.  If  the  sections  are  still  overstained,  place  them  in 
5  c.c.  of  distilled  water  to  which  3  drops  of  acetic  acid  have  been 
added.  Leave  for  5  minutes,  or  until  they  become  lighter  in 
color,  then  wash  in  several  changes  of  tap  water  until  they  have 
again  become  blue. 

10.  Remove  the  sections  from  the  water  and  transfer  them 
through  35,  50,  70,  85,  and  95  per  cent,  alcohol  successively, 
leaving  them  from  3  to  5  minutes  in  each,  and  lastly  transfer 
them  to  absolute  alcohol  for  10  minutes,  and  finally  to  carbol- 
xylol  for  10  minutes,  or  until  clear. 

11.  Select  one  or  two  of  the  best  sections  and  transfer  them  to 
the  center  of  a  clean  glass  slide.  After  straightening  them  out 
properly,  drain  off  the  excess  of  the  carbol-xylol,  and  before  the 
sections  can  become  dry,  add  a  drop  of  Canada  balsam.  Carefully 
lower  a  clean  cover-glass  (for  cleaning  see  memorandum  14,  chap, 
vi)  on  to  the  balsam.  There  should  be  just  sufficient  balsam  to 
spread  evenly  under  the  cover  without  exuding  around  the  edges. 

12.  Label,  stating  card  number,  name  of  the  preparation,  and 
other  data  that  it  is  desired  to  add  (see  chap,  vi,  i,  step  10). 

13.  Carry  one  of  the  pieces  of  stomach  prepared  in  Gilson 
through  the  same  treatment.  The  sections  should  be  transverse 
sections  of  the  stomach  wall. 

14.  Clean  up  all  dirty  glassware  immediately. 

MEMORANDA 

1.  The  Thinnest  Sections  are  not  always  the  best.  For  a  general 
view  of  an  organ,  large,  comparatively  thick  sections  are  usually  better; 
for  details  of  structure,  thin  sections. 

2.  Small  Pieces  of  Tissue  may  be  cemented  to  a  cork  if  too  small 
to  hold  conveniently  between  thumb  and  forefinger.  A  piece  of  stout 
copper  wire  is  heated  for  a  moment  in  the  flame  and  touched  to  a  bit 


Chapter  IV:  Simple  Section  Methods 


35 


of  paraffin.  As  the  paraffin  melts  transfer  drops  of  it  to  the  edge  of  the 
tissue,  which  has  been  previously  placed  on  the  cork.  The  paraffin-cools 
and  holds  the  tissue  fast. 

Another  and  better  method  of  handling  a  small  object  is  to  imbed  it 
in  a  piece  of  hardened  liver.  In  sectioning,  the  liver  as  well  as  the 
object  is  sliced,  but  they  readily  separate  when  placed  in  alcohol.  Beef 
liver  or  dog  liver  is  prepared  for  such  purposes  by  hardening  pieces  about 
5x2x2  cm.  in  size  in  95  per  cent,  alcohol  for  24  hours,  and  then  trans- 
ferring to  fresh  95  per  cent,  alcohol  until  needed.  When  much  hand 
sectioning  is  to  be  done,  a  supply  of  hardened  liver  should  be  kept  on 
hand.  Many  small  objects  may  be  held  between  pieces  of  pith,  and 
successfully  sectioned. 

3.  Well  Microtomes  (Fig.  27)  are  inexpensive  instruments  which  are 
used  for  simple  sectioning.  Such  a  microtome  consists  of  a  tube  in 
which  the  object  is  placed,  and  at  one  end  of 
which  is  a  plate  to  guide  the  razor.  The  other 
end  is  provided  with  a  screw,  wThich,  when  turned, 
pushes  the  contents  of  the  tube  above  the  plate, 
thus  making  it  possible  to  cut  sections  of  a  uni- 
form thickness.  The  object  to  be  cut  must  be 
firmly  fixed  in  the  well.  Such  tissues  as  kidney, 
liver,  spleen,  hard  tumors,  cartilage,  etc.,  may  be 
held  sufficiently  rigid  by  wedging  small  slabs  of 
carrot,  turnip,  pith,  or  hardened  liver  in  about 
them.  These  supporting  substances  must,  of 
course,  rest  squarely  against  the  bottom  of  the 
well.  Soft  tissues,  such  as  soft  tumors  or  brain, 
must  be  imbedded.  Three  parts  of  paraffin  and 
one  part  of  vaselin  melted  together  and  thoroughly 
mixed  makes  a  very  good  imbedding-mass  for  a 
well  microtome.  To  imbed,  warm  the  microtome 
slightly  and  fill  the  well  with  the  imbedding  mix- 
ture. Remove  all  liquid  from  the  surface  of  the 
tissue,  and  pass  it  below  the  surface  of  the  mixture 
just  as  it  begins  to  harden  around  the  edges, 
mass  has  become  cold  the  sections  are  cut  in  the  ordinary  way. 

4.  Temporary  Mounts  may  be  made  directly  from  water  after  staining 
by  using  glycerin  as  a  mounting-medium.  Transfer  the  section  to  the 
slide,  add  a  drop  or  two  of  glycerin,  and  a  clean  cover- glass. 


Fig.  27.— Well  Microtome. 


When  the  imbedding 


CHAPTER  V 
THE  PARAFFIN  METHOD:    IMBEDDING  AND  SECTIONING 

1.  From  70  per  cent,  alcohol  take  a  small  piece  of  intestine 
(6  mm.  long)  fixed  in  Gilson,  and  also  pieces  of  kidney  and  tongue, 
and  proceed  according  to  the  following  schedule.  Keep  accurate 
records  on  your  cards. 

2.  Ninety-five  per  cent,  alcohol,  30  to  45  minutes.  A  longer 
time  will  do  no  harm. 

3.  Absolute  alcohol,  45  minutes.  Before  transferring  to 
absolute,  remove  the  excess  of  95  per  cent,  alcohol  from  the 
object  by  touching  it  with  a  piece  of  blotting  paper  or  a  clean 
cloth. 

4.  Xylol,  2  hours  or  until  the  object  looks  clear.  It  may  be 
left  several  hours.  Rapidly  remove  all  excess  of  xylol  before 
proceeding  with  step  5,  but  do  not  allow  the  tissue  to  become  dry 
or  dull  looking. 

5.  Melted  paraffin  (melting-point  about  53°  C),  2  hours. 
The  object  may  be  left  an  hour  or  two  longer,  but  it  is  best  to 
avoid  as  much  as  possible  subjecting  tissues  to  an  elevated 
temperature.  Shift  its  position  in  the  paraffin  once  or  twice  to 
facilitate  penetration  of  the  latter. 

Cautions. — a)  Do  not  have  the  bath  too  hot.  Cooked  tissues 
are  worse  than  useless. 

b)  To  keep  material  clean,  it  is  well  to  have  a  false  bottom  of 
paper  in  the  vessel  containing  paraffin.  Make  this  by  swinging  a 
strip  of  white  paper  into  the  cup  so  that  the  loop  of  the  paper  is 
submerged  in  paraffin  and  the  ends  attached  on  either  side  to  the 
mouth  of  the  cup. 

6.  Prepare  paper  boxes  according  to  the  following  instructions: 

A  small  rectangular  block  of  wood  or  a  stick  with  a  flat  end  measuring 
approximately  15x20  mm.  is  used.  Cut  a  strip  of  stiff  paper  so  that  it 
measures  about  4x7  cm.  Place  the  flat  end  of  the  block  in  the  center  of 
the  paper  with  its  long  diameter  coinciding  with  the  long  diameter  of  the 

37 


38  Animal  Micrology 

paper.  Fold  the  narrow  side  margins  of  the  paper  up  along  the  sides  of 
the  block  first,  then  do  likewise  with  the  ends  of  the  paper.  Turn  the 
ears  which  have  been  formed  at  each  corner  back  over  what  is  to  be  the 
end  of  the  box,  and  then  fold  the  long  end  of  the  paper  back  to  hold 
the  ears  in  place,  and  also  to  make  the  end  of  the  box  of  the  same  height 
as  the  sides.  Manifestly,  any  size  of  box  may  be  made  by  varying  the 
size  of  the  block.  With  a  little  practice,  the  same  kind  of  box  may  be 
folded  without  the  use  of  a  wooden  block. 

7.  With  a  warm,  wide-mouthed  pipette  transfer  sufficient 
melted  paraffin  to  a  paper  box  to  cover  the  bottom,  then,  with 
warm  forceps,  remove  the  tissue  to  the  box.  Next,  fill  the  box 
with  melted  paraffin.  Orient  the  object  with  heated  needles  if 
necessary.  As  soon  as  the  paraffin  has  congealed  sufficiently  for 
the  surface  to  become  opaque,  cool  it  rapidly  by  plunging  it  into 
cold  water;  otherwise,  the  paraffin  will  crystallize  and  become 
unsuited  for  sectioning. 

Cautions. — a)  Tissues  must  be  oriented  (i.  e.,  placed  in  proper 
position  for  cutting)  while  the  paraffin  is  still  in  liquid  condition. 
Arrange  the  tissue  so  that  it  will  be  cut  at  right  angles  (trans- 
verse) or  parallel  to  the  surface  of  the  organ.  Avoid  oblique 
sections  as  they  are  very  puzzling.  For  present  purposes  of 
practice  cut  transverse  sections. 

b)  If  whitish-looking  patches  are  present  in  the  block  after 
imbedding  they  are  due  to  xylol  which  has  been  carried  over  into 
the  paraffin.  If  they  occur  in  the  immediate  vicinity  of  the  ob- 
ject, the  block  should  be  placed  in  the  bath  again  until  melted, 
and  the  object  be  reimbedded. 

c)  Be  sure  that  every  piece  of  tissue  is  marked  after  it  is  im- 
bedded. Tissues  are  sometimes  kept  in  paraffin  for  months  or 
even  years  before  they  are  finally  sectioned.  To  mark,  scratch 
the  number  of  the  record  card  in  the  paraffin,  or  better,  write  it 
on  the  paper  box  and  leave  the  box  in  place. 

CUTTING  SECTIONS 

8.  Study  the  paraffin  microtome  (e.  g.,  Fig.  28);  identify  the 
parts  and  learn  how  the  thickness  of  sections  is  controlled. 

9.  Proceed  with  the  block  of  paraffin  containing  the  intestine. 
Make  it  fast  to  the  carrying  disk  of  the  microtome  in  the  follow- 


Chapter  V:    The  Paraffin  Method 


39 


ing  manner:  Remove  the  disk  from  the  machine  and  by  means  of 
a  heated  steel  spatula  or  copper  wire  flattened  at  one  end,  melt- a 
small  chip  of  paraffin  on  to  it.  Likewise  warm  the  end  of  the 
paraffin   block  and  quickly  press  it  into  the  melted  paraffin  on 


Fig.  28.— Minot  Automatic  Rotary  Microtome. 

The  object  carrier  is  adjustable  in  three  planes  and  is  perfectly  rigid.  The  knife  carrier 
is  also  adjustable  and  extra  heavy  and  solid.  The  feed  is  controlled  by  an  adjustable  cam, 
giving  cuts  of  any  number  of  microns  in  thickness  from  1  to  25.  By  means  of  an  automati- 
cally closing  split-nut  the  carriage  is  returned  to  the  beginning  position  after  the  screw  is 
fed  out  the  entire  length. 

the  disk.  Cement  it  firmly  in  place  by  means  of  the  heated  wire 
or  spatula  and  cool  in  water. 

10.  With  a  sharp  scalpel  trim  the  free  end  of  the  block  so 
that  it  presents  a  perfectly  rectangular  outline  (however,  see 
caution  c).  The  length  should  exceed  the  breadth  by  at  least 
one-fourth. 

Cautions. — a)  In  trimming  do  not  cut  farther  back  than  the 
base  of  the  object.     This  leaves  a  wide  shoulder  for  support. 

b)  Leave  a  margin  of  about  2  mm.  around  the  object. 


40 


Animal  Micrology 


c)  To  avoid  reversing  sections  in  mounting,  it  is  frequently 
advantageous  to  have  the  imbedding  mass  trimmed  unsymmetri- 
cally.  The  edge  which  first  comes  in  contact  with  the  knife  is 
left  longer  than  the  opposite  edge.  One  may  thus  readily  dis- 
cover when  a  section  or  part  of  a  series  has  been  turned  over. 

11.  Mount  the  object  firmly  in  the  microtome.  It  should  just 
clear  the  knife.     The  flat  end-surface  of  the  paraffin  block  should 


Fig.  29.— Minot-Blake  Microtome,  designed  especially  for  cutting  thin  sections.  Manu- 
factured by  Buff  &  Buff  MYi/  Co.,  of  Boston,  Mass. 

be  parallel  to  the  edge  of  the  knife,  and  the  block  so  oriented 
that  in  cutting,  the  long  edge  will  meet  the  edge  of  the  knife 
squarely. 

12.  Place  the  knife  in  position  with  the  handle  to  the  side 
away  from  the  wheel  (if  a  rotary  microtome  is  used).  By  means 
of  the  adjusting  screws  tilt  the  cutting  edge  slightly  toward  the 
object  so  that  the  side  of  the  knife  will  not  remain  in  contact  with 


Chapter  V:    The  Paraffin  Method  41 

the  paraffin  block  after  a  section  has  been  cut.  If  the  knife  has 
a  flat  under  surface  it  requires  more  tilt  than  if  the  surface  is 
hollow  ground.  For  a  flat  under  surface  the  tilt  should  be  about 
9  degrees  from  the  perpendicular.  See  that  the  knife  is  held 
firmly  in  place. 

Caution. — The  knife  should  be  kept  in  its  case  when  not  in 
the  machine.     The  edge  is  very  easily  injured. 

13.  Set  the  regulator  so  that  the  microtome  will  cut  sections 
about  10  microns  thick.  A  micron  is  one-thousandth  of  a  milli- 
meter. 

14.  Unloose  the  catch  which  locks  the  wheel  and  revolve  the 
wheel  with  the  right  hand.  A  few  revolutions  should  bring  the 
block  of  paraffin  into  contact  with  the  knife.  As  each  new  section 
is  cut,  it  displaces  the  last  one  and  if  the  paraffin  is  of  the  proper 
consistency  unites  by  one  edge  with  the  displaced  section.  Thus 
a  ribbon  or  chain  is  formed.  When  the  ribbon  becomes  of  suffi- 
cient length  support  the  free  end  by  means  of  a  hair  brush  held 
in  the  left  hand.  To  prevent  breaking  the  ribbon  avoid  pulling 
it  taut.  A  silk  carrier  for  it  may  be  attached  to  the  machine  but 
there  is  little  need  for  such  after  one  has  acquired  a  little  skill  in 
supporting  it. 

Caution. — Never  bring  a  needle  or  other  hard  object  near  the 
edge  of  the  knife.  If  the  paraffin  does  not  ribbon  properly  con- 
sult the  table  at  the  end  of  this  chapter. 

15.  When  a  sufficient  number  of  sections  have  been  cut,  care- 
fully place  the  ribbon  on  a  piece  of  paper.  Protect  it  from 
draughts  of  air  which  will  carry  away  or  disarrange  the  sections. 

16.  Cut  the  ribbon  into  strips  of  such  length  that  they  may  be 
placed  in  successive  rows  one  above  the  other  under  the  cover- 
glass  that  is  to  be  used.  Mark  out  on  a  sheet  of  paper  the 
exact  size  of  the  cover-glass  so  that  there  can  be  no  mistake  in 
cutting  strips  of  the  proper  length.  A  margin  of  2  or  3  mm. 
should  be  allowed  for  the  cover. 

17.  Place  a  small  drop  of  albumen  fixative  on  a  clean  glass 
slide  (for  cleaning  see  memorandum  14,  chap,  vi),  and  spread  it 
evenly  over  the  surface,  except  the  end  which  is  to  bear  the  label 


42  Animal  Micrology 

(see  step  10,  chap.  vi).  With  a  clean  finger,  rnb  off  all  of  the 
fixative  that  can  be  easily  removed  so  that  only  a  very  thin  film 
remains. 

18.  Flood  the  slide  with  a  few  drops  of  distilled  water  until 
the  entire  surface  bearing  the  fixative  is  covered  by  a  thin  layer 
of  water,  but  do  not  put  on  sufficient  to  overflow  the  edge. 

19.  Take  up  the  first  strip  of  paraffin  ribbon  with  a  brush  or 
needle  and  float  it  onto  the  surface  of  the  water.  The  first  sec- 
tion of  the  series  should  be  in  the  upper  left-hand  corner,  but 
back  at  least  5  mm.  from  the  end  of  the  slide.  In  case  the  label 
is  to  be  placed  on  the  left  end  of  the  slide,  allowance  must  be 
made  for  it,  of  course.  Add  the  successive  strips  of  the  ribbon  in 
the  order  of  the  lines  of  a  printed  page  until  as  many  rows  are  in 
place  as  will  conveniently  lie  under  the  cover,  allowing  for  the 
proper  margins.  See  that  each  section  presents  the  same  aspect 
to  the  observer  as  its  predecessor  (see  10,  c). 

20.  Warm  the  slide  gently  by  holding  it  well  above  a  small 
flame  until  the  paraffin  flattens  out  and  becomes  free  from  wrinkles. 
Be  careful  not  to  melt  the  paraffin,  for  heat  sufficient  to  do  so  will 
render  the  albumen  useless.  It  is  safer  to  heat  the  slide  by 
placing  it  upon  the  warm  paraffin  oven  for  a  few  minutes,  instead 
of  holding  it  above  a  flame. 

21.  Drain  off  the  excess  of  water  and  set  the  slide  away  to  dry 
after  properly  numbering  it  with  your  glass-marking  pencil.  As 
the  water  evaporates  the  sections  are  drawn  down  tightly  into  the 
film  of  fixative.  The  slide  is  seldom  sufficiently  dried  under  six 
hours.  It  is  well  to  leave  it  twelve  hours ;  it  may  be  left  indefi- 
nitely. The  time  may  be  shortened  by  placing  a  few  thicknesses 
of  blotting  paper  under  the  slide  and  drying  it  on  the  paraffin 
oven.  Unless  the  slide  is  perfectly  dry  the  sections  will  float  off 
during  subsequent  treatment.  Take  precautions  to  prevent  par- 
ticles of  dirt  from  settling  upon  the  surface  of  the  sections.  This 
is  usually  accomplished  by  placing  the  slides  upon  some  kind  of  a 
rack  and  covering  them  with  a  bell- jar.  Prepare  several  other 
slides  in  the  same  manner  as  the  above  if  sufficient  of  the  ribbon 
remains. 


Chapter  V:    The  Paraffin  Method  43 

Note. — As  time  permits,  cut  the  other  sections  which  are 
imbedded  in  paraffin.  When,  as  in  the  present  case,  it  is  not 
necessary  to  have  a  complete  series  of  sections,  you  may  place 
fewer  sections  on  a  slide  and  use  smaller  covers. 

When  a  small  cover  is  to  be  used,  place  the  sections  at  the 
center  of  the  slide.  The  center  may  readily  be  determined  by 
drawing  the  outline  of  a  slide  on  a  card  and  connecting  the  oppo- 
site corners  of  the  figure  by  means  of  diagonal  lines.  When 
mounting,  place  a  slide  over  the  diagram ;  the  intersection  of  the 
diagonals  shows  the  center. 

At  this  point  the  student  should  make  a  careful  study  of 
Appendix  A  if  he  is  not  already  thoroughly  acquainted  with  the 
optical  principles  involved  in  microscopy. 

MEMORANDA 

1.  If  Paraffin  Becomes  Dirty  it  should  be  meited  and  filtered. 

2.  Oil  of  Cedar,  if  used  for  dealcoholization  before  imbedding,  should 
be  followed  by  at  least  two  changes  of  paraffin  or  the  paraffin  does  not 
thoroughly  replace  the  oil  and  the  object  is  likely  to  drop  out  of  the  sec- 
tions as  they  are  cut.  In  my  experience  this  is  the  commonest  difficulty 
which  beginners  encounter  if  they  use  cedar  oil  for  dealcoholization. 
For  this  reason  xylol  is  recommended  as  preferable  for  general  work. 

3.  Objects  Imbedded  in  Paraffin  may  be  preserved  in  that  form  indefi- 
nitely. It  is  one  of  the  most  convenient  ways,  in  fact,  of  preserving  mate- 
rial which  is  to  be  sectioned  in  paraffin. 

4.  Small  White  Objects,  if  not  stained  before  imbedding,  should  be 
tinged  with  a  dilute  solution  of  Bordeaux  red  to  facilitate  orientation. 
For  orientation  in  general  see  chap,  xvi,  memorandum  12. 

5.  With  Delicate  Tissues  it  is  necessary  that  the  transition  from  alco- 
hol to  clearer  be  gradual,  hence  it  is  best  to  add  the  clearer,  a  little  at 
a  time,  to  the  last  alcohol,  transferring  it  with  a  pipette  to  the  bottom  of 
the  alcohol. 

6.  The  Temperature  of  the  Laboratory  must  be  taken  into  account 
when  sectioning  in  paraffin.  In  summer  use  a  harder,  in  winter  a  softer, 
paraffin. 

7.  For  Thin  Sections  use  a  hard  paraffin,  for  thick  sections,  a  softer 
paraffin. 

8.  For  Valuable  Tissues  Which  Crumble  in  Paraffin  Alone  the  following 
somewhat  tedious  process  (Mark,  American  Naturalist  [1885],  p.  628) 
may  be  resorted  to.     Prepare  a  very  fluid  collodion  in  ether-alcohol  and 


44  Animal  Micrology 

coat  the  exposed  surface  of  the  object  immediately  before  cutting  each 
section.  If  the  collodion  leaves  a  shiny  surface  or  produces  a  mem- 
brane when  applied  to  the  paraffin,  it  is  not  thin  enough  and  must  be 
further  diluted  with  ether-alcohol.  Apply  the  collodion  with  a  brush 
with  all  excess  of  the  fluid  wiped  away  so  that  the  brush  is  just  moist. 
The  fluid  should  touch  only  the  face  of  the  block  in  which  the  object  is 
exposed.  After  applying,  wait  a  few  seconds  for  the  solution  to  dry 
before  cutting.    See  also  memorandum  9. 

9.  Johnson's  Paraffin-Asphalt-Rubber  Method  for  brittle  objects  is  a  very 
useful  one.  One  part  of  crude  India  rubber  cut  into  very  small  pieces 
is  mixed  with  ninety-nine  parts  of  hard  paraffin  which  has  previously 
been  melted  and  tinged  to  a  light  amber  color  with  a  small  amount  of 
asphalt  ("mineral  rubber").  The  mixture  is  then  subjected  to  a  tem- 
perature of  100°  C.  (not  higher)  for  24  to  48  hours,  or  left  in  a  paraffin 
oven  at  60°  C.  for  several  days.  Use  only  the  supernatant  fluid.  It  is 
allowed  to  cool  and  remain  cold  until  needed,  because  the  rubber  separates 
out  after  a  time  if  the  mixture  continues  melted.  Johnson  {Journal  of 
Applied  Microscopy,  Vol.  VI,  p.  2662)  recommends  it  as  even  better  than 
paraffin  for  all  kinds  of  work  for  which  paraffin  is  commonly  employed. 
Proceed  as  in  the  ordinary  method,  using  xylol  {not  cedar  oil)  for  dealco- 
holization  and  also  for  clearing  sections. 

10.  Keep  All  Parts  of  the  Microtome  clean  and  well  oiled  with  watch 
oil  or  pure  paraffin  oil  of  25  degrees.  The  instrument  should  be  covered 
when  not  in  use. 

11.  Keep  the  Microtome  Knife  Sharp.  It  should  receive  frequent  strop- 
pings.     For  sharpening  the  knife  two  hones  are  commonly  used. 

Honing.— li  the  knife  is  very  dull  it  is  first  honed  on  a  Belgian  yellow 
hone,  an  open-grained  stone  which  cuts  the  metal  of  the  knife  rapidly. 
The  surface  of  the  stone  is  kept  moist  with  filtered  kerosene  oil  or  lathered 
with  palm-oil  soap.  After  the  nicks  and  other  inequalities  of  the  edge  of 
the  knife  have  been  removed,  the  honing  is  best  finished  on  a  good  fine- 
grained blue-water  stone. 

In  honing  the  stone  is  laid  flat  on  the  table  with  its  end  toward  the 
operator  and  its  surface  properly  lubricated.  A  very  dull  knife  is  ground 
at  first  on  the  concave  side  only  until  it  developes  a  fine  "wire  edge" 
along  the  full  length  of  the  blade.  It  is  then  ground  on  each  side  alter- 
nately until  the  wire  edge  has  disappeared  completely.  In  grinding,  the 
knife  must  remain  flat  on  the  hone  and  pass  lightly  over  the  full  length 
of  the  surface,  edge  foremost  in  a  diagonal  direction  from  point  to  heel, 
although  itself  remaining  at  right  angles  to  the  long  axis  of  the  hone. 
The  honing  has  been  sufficient  when  all  nicks  and  wire  edges  have  disap- 
peared and  the  knife,  instead  of  catching  and  hanging  when  the  edge  is 


Chapter  V:    The  Paraffin  Method 


45 


drawn  lightly  across  the  ball  of  the  thumb,  freely  enters  the  moist  epi- 
dermis. Finally  the  blade  is  wiped  clean  with  a  soft  cloth,  great  care 
being  taken  not  to  injure  the  edge. 

Stropping. — A  broad  firm  strop  of  finest  calfskin  is  best.  It  should 
be  affixed  to  a  solid  back  so  that  it  will  not  spring  and  thus  round  off  the 
delicate  edge  of  the  knife. 

In  stropping,  the  motions  are  the  same  as  in  honing  (both  sides  of 
blade),  only  the  knife  passes  back  foremost  and  from  heel  to  point.  The 
blade  must  move  lightly  over  the  surface  of  the  strop  with  very  slight 
pressure  on  the  part  of  the  operator.  The  stropping  is  ordinarily  consid- 
ered sufficient  when  the  blade  will  cut  a  loose  hair  freely  along  every  part 
of  the  edge.  An  examination  under  a  low  power  of  the  microscope  should 
reveal  no  nicks  in  the  edge. 

12.  To  Remove  Pigments  and  to  Bleach  Osmic  and  Chromic  Acid  Materials 
a  3  per  cent,  solution  of  peroxide  of  hydrogen  frequently  is  sufficient. 
Tissues  left  too  long  in  this  liquid  macerate. 

Mayer's  chlorine  method  is  one  of  the  best  for  bleaching.  To  several 
crystals  of  chlorate  of  potash  in  a  glass  tube  a  few  drops  of  hydrochloric 
acid  is  added.  When  the  greenish  fumes  of  chlorine  appear,  add  from  5 
to  10  c.c.  of  50  per  cent,  alcohol.  The 
object,  which  in  the  meantime  has  been 
standing  in  70  per  cent,  alcohol,  is 
transferred  to  the  tube.  From  15  min- 
utes to  24  hours  are  required  for  bleach- 
ing, depending  upon  the  nature  of  the 
material.  It  is  well  to  suspend  the  ob- 
ject from  the  mouth  of  the  bottle.  Sec- 
tions on  the  slide  may  be  bleached  in  a 
few  minutes.  This  method  is  especially 
recommended  for  removing  natural  pigments  and  for  bleaching  osmic 
material. 

13.  Large  Objects  May  Be  Cut  in  Paraffin  better  with  a  slanting  knife 
than  with  a  square-set  one.  The  block  of  paraffin  must  be  trimmed 
to  a  three- sided  prism  with  its  most  acute  angle  farthest  from  the  object. 
A  sliding  microtome  is  used  ordinarily  and  the  block  of  paraffin  is  so 
oriented  that  the  knife  enters  at  the  sharpest  angle  of  the  prism.  Each 
section  as  cut  is  removed  with  a  brush. 

14.  Metal  "Ls"  Are  Frequently  Used  Instead  of  Paper  Boxes  for  mold- 
ing paraffin  blocks.  The  two  Ls  (Fig.  30)  may  be  placed  together 
on  a  small  glass  or  metal  plate  in  such  a  way  as  to  mold  blocks  of  any 
desired  size.  Before  pouring  the  melted  paraffin  in,  the  inner  walls  of 
the  metal  pieces  should  be  lightly  smeared  with  glycerin  so  that  the; 
block  of  paraffin  will  easily  separate  from  them  when  cool. 


Fig.  30.— Metal  |_s  for  molding 
imbedding  masses. 


46  Animal  Micrology 

DIFFICCJLTIES  LIKELY  TO  BE  ENCOUNTERED  IN  SECTIONING  IN 
PARAFFIN,  AND  THE  PROBABLE  REMEDY 

1.  Crooked  Ribbons. —  a)  Usually  caused  by  wedge-shaped  sections. 
Correct  by  trimming  the  block  of  paraffin  so  that  the  edge  which  strikes 
the  knife  first  and  the  edge  on  the  opposite  side  are  strictly  parallel. 
See  that  the  block  strikes  the  knife  exactly  at  right  angles. 

b)  The  paraffin  may  be  softer  at  one  end  of  the  block  than  at  the 
other.  This  can  only  be  corrected  by  imbedding  the  object  over  again 
in  a  homogeneous  paraffin. 

2.  The  Object  Makes  a  Scratching  Noise  on  the  Knife  or  Cuts  with  a  Gritty 
Feeling  and  the  sections  perhaps  crumble  and  tear  out  from  the  paraffin. 

a)  This  is  generally  caused  by  too  high  heating  of  the  object  while 
in  the  paraffin  oven.  Not  only  is  such  an  object  worthless  but  it  endan- 
gers the  edge  of  the  microtome  knife.  Correct  by  limiting  the  bath  in 
paraffin  to  the  minimum  time  necessary  for  a  proper  penetration  of  the 
object,  and  keeping  the  temperature  barely  above  the  melting-point  of 
the  paraffin. 

b)  The  fixing  reagent  has  formed  crystals  (e.g.,  corrosive  sublimate) 
which  have  not  been  thoroughly  washed  out. 

See  also  5. 

3.  The  Sections  Wrinkle  or  Jam  Together;  the  object  itself  may  be  com- 
pressed before  the  knife.  This  is  a  serious  fault  because  the  arrange- 
ment of  the  parts  of  a  tissue  are  greatly  deranged.  It  may  be  due  to 
various  causes. 

a)  The  microtome  knife  may  be  dull.  Examine  the  knife  and  sharpen 
it  if  necessary. 

b)  The  paraffin  may  be  too  soft.  To  remedy  this  defect  employ  one 
or  more  of  the  following  means:  (1)  cool  the  paraffin  block  in  water; 
(2)  cut  the  sections  in  a  cooler  room;  (3)  cut  the  sections  thicker;  (4)  re- 
imbed  in  harder  paraffin.  If  sections  are  not  too  badly  wrinkled  they 
may  be  flattened  out  by  warming  on  water  as  directed  in  steps  18-20. 

c)  A  possible  reason  is  that  the  tilt  of  the  knife  is  insufficient  (see 
step  12). 

d)  The  edge  of  the  knife  may  be  smeared  with  a  layer  of  paraffin. 
Clean  the  edge  with  a  cloth  moistened  in  xylol. 

4.  The  Sections  Roll  and  Refuse  to  Ribbon.  —  This  is  one  of  the  most 
exasperating  of  all  defects.  If  the  sections  are  not  tightly  curled 
they  frequently  unroll  when  placed  on  warm  water  (step  18).  Various 
mechanical  devices  have  been  constructed  to  prevent  this  evil,  but  most 
of  them  are  impractical.  Sometimes  when  a  section  begins  to  roll,  if  the 
edge  is  held  down  by  means  of  a  flat-pointed  hair  brush,  the  curling  can 
be  overcome.  If  a  ribbon  can  once  be  started  the  difficulty  is  frequently 
corrected.    The  sections  should  be  cut  rapidly. 


Chapter  V:    The  Paraffin  Method  47 

a)  The  commonest  cause  of  rolling  is  the  hardness  of  the  paraffin. 
This  may  sometimes  be  remedied  by  one  or  more  of  the  following  means-! 
(1)  warming  the  knife  with  the  breath;  (2)  cutting  in  a  warmer  room; 
(3)  placing  a  lamp  or  burner  near  the  imbedded  object;  (4)  warming  the 
knife  very  carefully  by  holding  the  back  on  a  warm  paraffin  bath; 
(5)  cutting  the  sections  thinner;  (6)  reimbedding  the  object  in  softer  par- 
affin. 

b)  The  tilt  of  the  knife  may  be  too  great  ( step  12 ). 

c)  The  knife  may  be  dull. 

5.  The  Sections  Split  Longitudinally  or  Are  Crossed  by  Parallel  Scratches. — 
a)  Look  for  a  nick  in  the  edge  of  the  knife.  Cut  in  a  new  place  on  the 
knife  or  sharpen  it. 

b)  A  bit  of  grit  may  have  gotten  into  the  object  or  the  paraffin. 
Keimbed  after  carefully  cleaning  the  object  in  the  clearing  fluid. 

c)  Tissues  may  contain  hard  substances  (lime  salts,  silica,  crystals 
precipitated  from  fixing  reagents)  which  have  been  imperfectly  washed 
out.  It  is  best  to  take  an  entirely  new  piece  of  tissue  in  which  these 
defects  do  not  exist. 

d)  The  tilt  of  the  knife  may  be  too  great  (step  12). 

e)  The  object  may  be  too  large  to  cut  in  paraffin.  Try  smaller  pieces 
of  tissue  or  use  the  celloidin  method. 

6.  The  Knife  Scrapes  or  Rings  as  It  Passes  Back  over  the  object  after 
having  cut  a  section. 

a)  This  is  sometimes  caused  by  a  knife  with  either  too  great  or  too 
little  tilt  (step  12). 

b)  The  object  may  be  too  tough  or  hard  to  cut  in  paraffin  without 
springing  the  edge  of  the  knife  ( see  7  6). 

7.  The  Sections  Vary  in  Thickness;  the  machine  cuts  one  thick  and  one 
thin  or  misses  a  section. 

a)  This  may  be  caused  by  the  imperfect  mechanical  construction  of 
the  machine.  Old  machines  in  which  the  parts  are  worn  are  especially 
liable  to  this  defect.  It  may  be  remedied  to  some  extent  by  tightening 
up  the  parts  of  the  machine. 

b)  The  object  may  be  too  hard  for  the  knife  to  cut  and,  as  a  conse- 
quence, the  edge  of  the  knife  springs.  When  tough  or  hard  objects  must 
be  cut,  use  an  old  microtome  knife  or  a  sectioning  razor.  See  if  there  is 
not  some  means  of  softening  such  a  tissue  without  obscuring  the  micro- 
scopical structures  sought. 

c)  Either  too  great  or  too  little  tilt  may  cause  the  defect  (step  12). 

d)  See  that  the  disk  bearing  the  object  is  securely  clamped  in  the 
machine. 

8.  The  Object  Crumbles  or  Drops  out  of  the  Paraffin  as  Cut.  —  It  has 
probably    been    insufficiently    penetrated    by    paraffin.     Some  of  the 


48  Animal  Micrology 

following  precautions  may  prevent  tfre  defect:  (1)  Leave  the  object 
in  the  paraffin  bath  longer.  (2)  See  that  it  is  entirely  free  from  the  de- 
alcoholizing  fluid  before  placing  it  into  the  melted  paraffin.  Objects 
which  have  been  immersed  in  cedar  oil  are  particularly  subject  to  this 
defect.  For  this  reason  xylol  is  better  than  cedar  oil  for  dealcoholization 
in  general  work.  (3)  If  the  object  is  impervious  to  paraffin  or  very  friable, 
as  are  many  ova,  some  other  method  must  be  tried.  Consult  memoranda 
8  and  9;  see  also  the  celloidin  method  (chap,  vii)  or  the  combination 
celloidin -paraffin  method  (chap,  vii,  memorandum  8). 

9.  The  Ribbon  Twists  or  Curls  about  or  Clings  Closely  to  the  Side  of  the 
Knife. — This  is  due  to  the  electrification  of  sections.  If  the  fault  is 
excessive  it  is  best  to  postpone  the  cutting  until  the  atmospheric  con- 
ditions have  changed. 


CHAPTER  VI 

THE  PARAFFIN  METHOD:  STAINING  AND  MOUNTING 

I.     STAINING  WITH  HEMATOXYLIN 

Place  enough  of  the  following  reagents  in  tall  stender  dishes 
or  Coplin  staining-jars  to  cover  the  slides  lengthwise,  up  beyond 
the  sections  affixed  to  them:  xylol,  carbol-xylol,  absolute,  95,  70, 
50,  35  per  cent,  alcohols  respectively,  clear  water,  acid  alcohol, 
and  for  washing  out  the  acid  alcohol  in  the  case  of  hematoxylin 
preparations,  a  separate  jar  of  70  per  cent,  alcohol  to  which  a 
few  drops  of  a  0.1  per  cent,  aqueous  solution  of  bicarbonate  of 
soda  has  been  added.  Arrange  these  reagents  in  a  row  in  the 
order  named  with  the  exception  of  the  acid  alcohol  and  its  accom- 
panying alkaline  alcohol  wash  of  70  per  cent,  alcohol,  which  may 
be  placed  immediately  back  of  the  ordinary  70  per  cent,  alcohol. 
Put  a  little  vaselin  along  the  upper  edges  of  the  jars  containing 
absolute  alcohol,  xylol,  and  carbol-xylol  and  press  the  cover  down 
tightly  to  prevent  evaporation  or  the  entrance  of  moisture. 

In  like  manner  place  in  Coplin  staining-jars  (tall  stenders  will 
answer)  a  supply  of  Delafield's  hematoxylin  diluted  one-half  with 
distilled  water,  eosin,  Lyons  blue,  borax-carmine,  Bordeaux  red,  and 
solutions  A  and  B  for  the  iron-hematoxylin  method.  Arrange 
these  stains  in  a  row  back  of  the  alcohol  series. 

1.  Remove  the  paraffin  from  the  sections  of  intestine  (see  last 
lesson)  by  placing  the  slides  in  xylol  (turpentine  will  answer) 
for  10  or  15  minutes.  The  process  may  be  hastened  by  first 
gently  warming  the  slide  until  the  paraffin  begins  to  melt. 

2.  Remove  the  xylol  from  the  sections  by  transferring  the 
slides  to  absolute  alcohol  for  1  minute. 

3.  Pass  the  slides  through  the  alcohols  (95,  70,  50,  and  35 
per  cent. )  leaving  them  for  a  half-minute  in  each. 

4.  Remove  to  Delafield's  hematoxylin  for  10  to  30  minutes  or 
until  stained  a  pronounced  blue. 

5.  Wash  in  water  for  5  minutes. 

49 


50  Animal  Micrology 

6.  Pass  the  slides  up  through  the  series  of  alcohols  to  70  per 
cent.,  leaving  them  about  half  a  minute  in  each  alcohol. 

7.  Dip  each  slide  for  from  thirty  seconds  to  five  minutes  into 
the  acid  alcohol  until  the  sections  are  of  a  reddish  hue,  then  rinse 
them  in  70  per  cent,  alkaline  alcohol  until  the  blue  color  is 
restored.  This  last  alcohol  must  be  kept  very  slightly  alkaline 
through  the  occasional  addition  of  a  few  drops  of  a  0.1  per  cent, 
solution  of  bicarbonate  of  soda  (see  memorandum  10).  The  alka- 
line alcohol  may  be  omitted  when  other  than  hematoxylin  stains 
are  used  as  its  purpose  is  merely  to  restore  the  blue  color  of  the 
latter. 

8.  Pass  the  slides  through  95  per  cent,  alcohol  (1  minute), 
absolute  alcohol  (3  minutes),  into  carbol-xylol  for  5  minutes  or 
until  clear. 

9.  Carefully  drain  off  all  excess  of  the  clearer,  wipe  the  under 
side  of  a  slide  and  lay  it  down  flat  with  the  sections  uppermost. 
Put  a  few  drops  of  thin  balsam  on  the  sections  near  one  end. 
Take  up  a  clean  cover-glass  and  holding  it  by  the  edges  between 
the  thumb  and  first  finger  of  one  hand,  lower  it  upon  the  balsam  by 
bringing  one  end  into  contact  with  the  slide  near  the  balsam,  and 
supporting  the  other  end  by  means  of  a  needle  held  in  the  free 
hand.  Lower  the  cover  slowly  so  that  as  the  balsam  spreads  no 
air  bubbles  will  be  inclosed  under  the  glass.  If  a  slide  is  tilted 
a  little  and  allowed  to  remain  in  that  position  small  bubbles  will 
frequently  work  out  unaided.  They  may  sometimes  be  removed 
by  pressing  gently  above  them  with  the  handle  of  a  needle  and 
gradually  working  them  to  the  edge  of  the  cover-glass.  Keep 
the  slide  in  a  horizontal  position  until  the  balsam  hardens. 

Caution. — Do  not  allow  the  sections  to  become  dry  before 
adding  the  balsam  and  cover. 

10.  Attach  the  permanent  label.  It  should  contain  at  least 
the  following  data:  the  number  of  the  record  card;  the  name 
of  the  tissue;  the  kind  of  section  (plane  of  section,  thickness, 
etc.),  if  one  of  a  series  the  number  of  the  slide  in  the  series 
and  the  number  of  the  first  and  last  section  on  the  slide;  the 
date,  and  if  desired  the  name  or  the  initials  of  the  preparator. 


Chapter  VI:    The  Paraffin  Method  51 

If  the  operator  is  accustomed  to  manipulate  the  fine  adjustment 
of  his  microscope  with  the  left  hand  and  the  slide  on  the  stage 
with  the  right,  it  is  best  to  have  the  label  on  the  right  end  of 
the  slide;  if  not,  then  on  the  left. 

Note. — Prepare  four  slides  each  of  the  other  objects  which 
have  been  imbedded.  Stain  and  mount  one  of  each  kind  as  you 
did  the  intestine,  and  also  one  of  each  kind  in  the  same  way, 
only  substitute  borax-carmine  for  the  hematoxylin.  The  borax- 
carmine  may  require  6  to  12  hours  for  staining.  Preserve  the 
others  for  double  staining. 

As  time  permits  prepare  and  section  the  other  tissues  which 
were  fixed  in  alcohol  and  Gilson.  After  you  have  had  the 
preliminary  practice  in  double  staining,  stain  and  mount  these  as 
you  prefer. 

II.  DOUBLE  STAINING  IN  HEMATOXYLIN  AND  EOSIN 

1.  Proceed  according  to  the  regular  schedule  with  one  each  of 
the  slides  reserved  above,  and  stain  in  Delafield's  hematoxylin. 

2.  Wash  the  sections  in  water,  and  proceed  farther  according 
to  the  regular  schedule  to  95  per  cent,  alcohol. 

3.  Transfer  the  slide  to  the  eosin  stain  for  10  to  30  seconds, 
and  after  rinsing  again  in  95  per  cent,  alcohol,  place  it  in  absolute 
alcohol. 

4.  Clear  in  carbol-xylol  and  mount  in  balsam. 

Note. — The  sections  should  show  both  the  blue  stain  (in  nuclei)  and 
the  red  stain  (in  cytoplasm)  when  examined  under  the  microscope.  If 
either  is  too  dense  or  too  light,  make  a  note  of  the  fact  and  vary  the  time 
accordingly  when  staining  other  sections  by  this  method. 

III.  DOUBLE  STAINING  IN  CARMINE  AND  LYONS  BLUE 

1.  Pass  the  remaining  reserved  slides  through  xylol  and  the 
alcohols,  descending  to  35  per  cent,  alcohol. 

2.  Stain  in  borax-carmine  for  from  30  minutes  to  several  hours, 
until  the  sections  are  well  colored. 

3.  Rinse  in  water  or  35  per  cent,  alcohol,  and  pass  the  sections 
up  through  the  alcohols  to  95  per  cent.  If  the  sections  are 
deeply  stained,  however,  remove    the  excess   of  stain  with  acid 


52  Animal  Micrology 

alcohol  (a  few  seconds)  when  the   sections  are  in  70  per  cent, 
alcohol. 

4.  Stain  for  10  to  20  seconds  in  Lyons  blue.  It  is  very  easy 
to  overstain  with  this  dye. 

5.  Rinse  in  95  per  cent,  alcohol,  and  transfer  the  sections  to 
absolute  alcohol  (3  minutes),  clear  in  carbol-xylol,  and  mount  in 
balsam. 

IV.  STAINING  WITH  HEIDENHAIN'S  IRON-ALUM  HEMATOXYLIN 

This  stain  is  very  valuable  in  the  study  of  cell  division  and  in 
determining  the  finer  structure  of  the  nucleus.  The  iron-alum 
acts  as  a  mordant,  preparing  the  tissue  for  the  action  of  the 
hematoxylin. 

1.  Prepare  two  sets  of  sections  of  intestine,  testis,  or  ovary, 
bladder,  pancreas,  and  stomach.  The  sections  should  not  be  over 
6  or  7  microns  in  thickness.  Preserve  one  set  for  double 
staining. 

2.  Pass  the  slides  bearing  the  sections  through  xylol,  absolute 
alcohol,  yo  per  cent,  alcohol,  ana-tnonec  directly  into  water.  thr*»    H» 

3.  Transfer  from  water  to  the  iron-alum,  and  allow  this  solu- 
tion to  act  for  from  6  to  8  hours. 

4.  Rinse  in  water  5  minutes. 

5.  Stain  in  the  0.5  per  cent,  hematoxylin  24  to  36  hours.  If 
a  trace  of  the  iron-alum  remains  in  the  sections  the  hematoxylin 
will  tarn  black.  This,  however,  does  not  impair  its  power  of 
staining. 

6.  Rinse  in  water  5  minutes. 

7.  Place  the  sections  into  iron-alum  again,  which  will  now 
extract  the  excess  of  stain.  The  time  required  for  proper  differ- 
entiation varies  with  the  kind  of  tissue  and  the  fixing  agent  that 
has  been  used.  From  10  to  30  minutes  is  usually  sufficient, 
though  no  definite  time  limit  can  be  set.  Remove  the  slide  from 
the  iron-alum  from  time  to  time  and  inspect  it.  When  the  sec- 
tions become  of  a  dull-grayish  hue  the  decolorization  is  usually 
sufficient.  If  very  accurate  results  are  necessary,  the  slide  should 
be  removed  from  the  iron-alum  frequently  and  examined  under 


Chapter  VI:    The  Paraffin  Method  53 

the  microscope.  When  in  a  dividing  cell  the  chromosomes  become 
sharply  denned,  the  decolorization  should  be  stopped. 

8.  Wash  in  several  changes  of  water  for  2  to  3  hours.  If  any 
of  the  iron-alum  is  left  in  the  sections  the  color  will  fade  later. 

9.  Wipe  off  the  excess  of  water,  transfer  the  slide  to  95  per 
cent,  alcohol,  followed  by  absolute  alcohol  and  carbol-xylol. 

10.  Mount  in  balsam. 

Note. —  Iron  hematoxylin  is  perhaps  the  one  most  important  stain  in 
use  today.  The  student  should  practice  the  method  until  he  has 
mastered  it. 

It  is  better  though  not  absolutely  essential  that  the  stain  be  "  ripe." 
If  the  stain  is  to  be  simply  for  general  histological  instead  of  cytological 
work,  the  baths  may  be  curtailed  considerably.  For  example,  immersion 
for  30  minutes  in  the  iron  solution,  then  for  45  minutes  in  the  stain  fol- 
lowed by  very  brief  differentiation  in  the  iron  solution,  yields  a  good 
general  preparation  but  the  finer  details  of  cell  structure  (centrosome, 
etc.)  are  not  brought  out. 

V.  BORDEAUX  RED  AND  IR^N-ALUM  HEMATOXYLIN 

■J 

Use  the  sections  which  were  reserved  for  this  method.     The 

method  is  identical  with  the  one  just  outlined,  except  that  between 

step  2  and  step  3  the  following  directions  should  be  inserted:  2a, 

transfer  the  sections  from  water    to  Bordeaux  red  for  2  hours 

(12  hours  will  do  no  harm),  then  wash  them  in  water  and  proceed 

to  step  3. 

Note. — Before  proceeding  further,  kill  a  female  cat  or  rabbit  to 
secure  tissues  for  the  celloidin  method  and  to  correct  failures  in  the 
paraffin  method.  In  addition  to  the  tissues  specified  before,  prepare 
{fix  in  Gilson)  pieces  of  tendon,  cartilage,  spleen,  lymph  gland,  pancreas, 
and  salivary  glands.  (If  the  reagents  are  at  hand  and  time  permits, 
the  student,  indeed,  might  advantageously  prepare  a  number  of  tissues 
according  to  the  methods  indicated  in  Appendix  C.)  Fix  the  ovary  in 
Gilson,  and  reserve  it  for  the  paraffin  method  for  delicate  objects.  Fix 
parts  of  the  brain  and  cord  in  Erlicki  and  in  formalin  as  previously 
indicated,  and  place  bits  of  muscle  in  which  nerves  terminate  plentifully 
(e.g.,  intercostals)  in  formalin.  Larger  pieces  (up  to  2  cm.)  may  be 
used  of  such  tissues  as  are  to  be  imbedded  in  celloidin .  Bear  in  mind 
that  the  larger  the  tissue  the  longer  must  it  be  left  in  the  different 
reagents.  Select  the  necessary  parts  of  the  digestive  tract  to  prepare 
longitudinal  sections  in  celloidin  from  esophagus  to  stomach  and  from 
stomach  to  intestine.  As  soon  as  possible  begin  the  preliminary  steps 
in  the  celloidin  method  (chap,  vii)  so  that  there  may  be  no  loss  of  time. 


54  Animal  Micrology 

Prepare  a  piece  of  intestine  for  staining  in  bulk  {see  vi).  It  should  be 
placed  in  the  stain  after  thoroughly  washing  out  the  fixing  reagent. 
Preserve  parts  of  it  to  cut  in  celloidin.  Remove  the  lower  jaw,  and 
prepare  it  for  decalcification  of  teeth  as  indicated  in  chap.xi.  Likewise 
prepare  pieces  of  femur  and  of  tarsal  bone  for  sectioning  {chap.  xi). 

VI.  STAINING  IN  BULK  BEFORE  SECTIONING 
It  is  sometimes  desirable  to  stain  objects  before  sectioning. 
The  method  is  a  slow  one,  and  requires  stains  which  penetrate 
evenly  and  thoroughly.  Various  preparations  of  carmine  and 
cochineal  give  the  best  satisfaction,  although  several  hematoxylin 
stains  are  also  frequently  used  in  this  way.  It  is  best  to  stain 
immediately  after  fixing  and  washing  out,  before  the  object  has 
been  carried  into  higher  alcohols.  In  general,  it  is  advisable  to 
section  tissues  and  stain  on  the  slide,  because  the  staining  can  be 
controlled  more  effectually.  Use  the  piece  of  intestine  already 
prepared  (see  note  above). 

1.  After  fixing  in  Grilson  and  thoroughly  washing  out  in 
water,  place  the  tissue  in  borax-carmine  for  24  hours. 

2.  Wash  in  35  per  cent,  alcohol  for  5  minutes. 

3.  Fifty,  70,  95  per  cent,  alcohols,  30  minutes  each. 

4.  From  this  point  proceed  through  absolute  alcohol,  xylol, 
and  imbedding,  sectioning,  and  mounting  precisely  as  in  the 
general  paraffin  method,  except  that  after  the  sections  have  been 
freed  from  paraffin  in  xylol,  do  not  mount  immediately  in  balsam, 
but  first  transfer  the  slide  back  into  absolute  alcohol,  and  thor- 
oughly wash  it  in  order  to  remove  the  glycerin  from  the  fixative 
and  so  prevent  cloudiness  of  the  final  mount.  From  alcohol  the 
slide  is  passed  through  xylol,  or  carbol-xylol,  and  mounted  in  the 
usual  way. 

Note. — When  borax -carmine  or  Delafield's  hematoxylin  is  used  as 
the  stain  for  an  entire  object,  the  preparation  usually  needs  to  be 
decolorized  with  acid  alcohol.  This  may  be  deferred,  however,  until 
after  the  object  is  sectioned. 

VII.     PARAFFIN  METHOD  FOR  DELICATE  OBJECTS 

To   prevent   the   distortion   of   delicate   objects  which  are  to 

be  sectioned  in  paraffin  the  transition  of  the  material  from  one 

reagent  to  the  other  must  be  very  gradual  and  the  heat  be  mini- 


Chapter  VI:    The  Paraffin  Method  55 

mized.    Observe  the  following  modifications  of  the  general  method 
and  prepare  pieces  of  ovary  which  have  been  fixed  in  Gilson. 

1.  Pass  the  object  in  the  usual  manner  up  through  the  series 
of  alcohols  to  absolute.  It  is  sometimes  necessary  to  use  a  more 
closely  graded  series  of  alcohols  if  the  object  be  very  delicate. 

2.  From  the  absolute  alcohol,  pass  to  a  mixture  of  absolute 
alcohol  two-thirds  and  chloroform  one-third;  gradually  add  more 
chloroform  until  at  the  end  of  an  hour  the  mixture  is  at  least 
two-thirds  chloroform. 

3.  Transfer  to  pure  chloroform  for  30  minutes. 

4.  Add  melted  paraffin  little  by  little  during  the  course  of  an 
hour  or  two  (24  hours  will  do  no  harm),  until  the  chloroform  will 
hold  no  more  in  solution. 

5.  Transfer  the  object  to  pure  melted  paraffin  in  a  small  ves- 
sel on  the  paraffin  oven  for  10  to  20  minutes,  changing  the  par- 
affin once.     Imbed  in  the  usual  way. 

6.  Cut  the  sections  about  7  microns  thick.     Mount  and  stain 

some  in  Delafield's  hematoxylin  and  eosin,  and  others  in  iron- 

hematoxylin   and  Bordeaux   red,   according    to    the    directions 

already  given  for  these  methods. 

Note. — For  very  sensitive  objects  Schultz's  dehydrating  apparatus 
(to  be  obtained  from  dealers)  may  be  used.  It  consists  of  a  tube  within 
a  tube,  each  having  the  lower  end  covered  by  an  animal  membrane.  The 
tubes  are  suspended  in  the  neck  of  a  much  larger  bottle  which  contains 
95  per  cent,  alcohol.  The  object  is  placed  in  the  inner  tube  and  both 
tubes  filled  with  water.  When  suspended  in  the  alcohol,  a  very  gradual 
hardening  or  dehydration  of  the  object  takes  place  as  the  alcohol  slowly 
diffuses  through  the  membrane.  Sometimes  it  is  necessary  to  use  only 
one  tube,  and  in  such  a  case  the  hardening  proceeds  more  rapidly. 

MEMORANDA 

1.  In  Passing  from  One  Liquid  to  Another,  one  corner  of  the  slide-bear- 
ing sections  should  first  be  touched  by  blotting  paper  to  remove  any 
excess  of  the  liquid  last  used.  This  is  especially  necessary  in  trans- 
ferring from  absolute  alcohol  to  xylol,  or  from  95  per  cent,  to  absolute 
alcohol. 

2.  Sections  Once  Placed  in  Turpentine  or  Xylol  for  the  removal  of  par- 
affin must  never  in  any  subsequent  step  be  allowed  to  become  dry. 
Particular  care  must  be  taken  to  prevent  sections  from  drying  out  after 
removing  them  from  xylol  to  mount  in  balsam  because  the  xylol  evapo- 
rates rapidly.  If  the  sections  become  dry  the  preparation  is  usually 
rendered  valueless. 


56  Animal  Micrology 

3.  Xylol  Used  for  Removing  Paraffin  should  be  kept  in  a  jar  separate  from 
that  which  contains  xylol  for  clearing  before  mounting  and  it  should  be 
changed  occasionally  because  it  tends  to  become  saturated  with  paraffin. 

4.  Sections  Not  over  io  Microns  Thick  may  be  plunged  directly  from  95 
per  cent,  alcohol  into  an  aqueous  medium  and  vice  versa.  If  sections 
are  over  10  microns  thick  it  is  better  to  put  them  through  the  complete 
series  of  alcohols.  With  thick  sections  diffusion  is  less  rapid,  and  too 
abrupt  a  change  from  one  fluid  to  another  may  produce  distortions  or 
wrench  the  sections  loose  from  the  slide. 

5.  To  Avoid  Rubbing  Sections  off  the  Slide,  hold  the  slide  with  one  end 
toward  the  light  before  wiping  it  and  glance  obliquely  along  the  surface. 
The  shiny  side  is  the  one  to  wipe. 

6.  The  Series  of  Alcohols  and  Stains  ordinarily  may  be  used  a  number 
of  times  without  replenishing.  When  the  alcohols  become  very  much 
discolored  or  the  stains  cloudy  they  should  be  renewed.  Alcohols  should 
not  be  used  too  often,  however,  as  they  soon  accumulate  particles  of  dirt 
which  settle  upon  the  sections  and  render  preparations  unsightly. 

7.  Absolute  Alcohol  must  be  kept  free  from  water.  It  may  be  tested 
from  time  to  time  by  mixing  a  few  drops  with  a  little  turpentine.  If  the 
mixture  appears  milky  the  alcohol  contains  a  harmful  amount  of  water 
and  should  be  renewed. 

8.  Two  Slides  Placed  Back  to  Back  can  be  handled  as  readily  as  a  single 
slide  in  passing  through  the  various  liquids. 

9.  Gentle  Agitation  of  a  Slide  in  any  liquid  facilitates  the  action  of  the 
liquid.     Observe  this  precaution  especially  with  absolute  alcohol. 

10.  For  Washing  Sections  after  Staining  in  Hematoxylin  tap  water  is 
preferable  to  distilled  water  because  it  is  usually  slightly  alkaline. 
When  acid  alcohol  is  used  to  decolorize  sections  stained  in  hematoxylin, 
the  sections  should  be  washed  in  70  per  cent,  alcohol  rendered  alkaline 
by  the  addition  of  a  few  drops  of  0.1  per  cent,  solution  of  bicarbonate  of 
soda.  The  alkali  neutralizes  the  acid  and  restores  the  bluish-purple 
color  to  the  section;  it  also  renders  the  blue  color  more  permanent.  If 
too  much  of  the  soda  is  added  the  color  will  be  a  hazy  disagreeable  blue. 

11.  To  Obtain  a  More  Precise  Stain  with  Delafield's  hematoxylin  it  is 
well  to  dilute  it  with  three  or  four  times  its  bulk  of  distilled  water. 
The  sections  must  be  left  in  this  solution  a  correspondingly  longer  time. 
Sections  stained  in  this  way  may  not  require  treatment  with  acid  alcohol. 
Most  workers,  however,  prefer  to  overstain  and  decolorize. 

12.  The  Length  of  Time  Required  for  Staining  Different  Tissues  is  exceed- 
ingly variable.  Upon  removal  from  the  stain  after  rinsing,  if  the  sections 
are  insufficiently  colored,  put  them  back  into  the  stain  and  examine  from 
time  to  time  until  they  are  properly  stained  (30  minutes  to  24  hours). 

13.  If  Objects  Refuse  to  Stain  it  is  usually  due  to  one  of  the  follow- 
ing causes:    (a)  The  fixing  agent  has  not  been  sufficiently  washed  out. 


Chapter  VI:    The  Paraffin  Method  57 

This  is  a  frequent  cause  of  poor  staining,  (b)  The  fixation  has  been 
poor.  The  success  of  a  preparation  depends  largely  upon  proper  fixation: 
in  most  cases,  (c)  The  stain  is  at  fault.  Hematoxylin  will  not  stain 
properly  until  ripe  (see  Hematoxylin,  page  20).  Many  stains,  especially 
the  anilins,  deteriorate  and  must  be  replaced,  (d)  Certain  stains  will 
not  follow  some  fixing  agents.  This  can  be  remedied  only  by  using  a 
different  stain  or  by  fixing  tissues  in  a  different  fluid.  The  hematoxy- 
lins and  carmines  are  applicable  after  a  very  large  variety  of  fixing 
agents,  (e)  The  paraffin  has  been  insufficiently  removed  from  the  sec- 
tions. This  may  be  corrected  by  dissolving  off  the  cover-glass  in  xylol 
and  after  thoroughly  removing  all  paraffin,  restaining  and  mounting 
the  sections  again  in  the  ordinary  way. 

14.  Use  Only  Clean  Slides  and  Covers. — Always  grasp  a  slide  or  a 
cover  by  its  edges  to  avoid  soiling  its  surface.  All  cloudiness  (seen  by 
looking  through  the  glass  toward  some  dark  object)  must  be  removed. 
For  wiping  slides  and  covers,  a  piece  of  cloth  which  does  not  readily 
form  lint  should  be  used.  Slides  may  often  be  cleaned  after  simply 
dipping  them  into  alcohol  or  into  alcohol  followed  by  water.  If  this 
treatment  is  insufficient,  place  them  for  several  hours  into  equal  parts  of 
hydrochloric  acid  and  95  per  cent,  alcohol,  keeping  them  well  separated 
so  that  the  liquid  may  act  on  the  entire  surface  of  each.  Then  rinse  them 
in  water  and  place  them  in  ether-alcohol.  It  is  well  to  keep  a  stock 
supply  of  such  slides  and  cover- glasses  in  ether-alcohol. 

To  clean  a  cover-glass  grasp  it  by  the  edges  in  one  hand,  cover  the 
thumb  and  first  finger  of  the  other  hand  with  the  cleaning  cloth  and 
rub  both  surfaces  of  the  glass  at  the  same  time.  To  avoid  breaking  the 
cover,  keep  the  thumb  and  finger  each  directly  opposite  the  other.  A 
large  cover-glass  may  be  cleaned  by  rubbing  it  between  two  flat  blocks 
which  have  been  wrapped  with  cleaning  cloths. 

To  clean  slides  which  have  been  used,  if  balsam  mounts,  warm  and 
place  in  xylol  or  turpentine  to  dissolve  off  the  covers.  Put  the  slides 
and  covers  into  separate  vessels  and  leave  them  for  a  few  days  in  the 
following  cleaning  mixture: 

Potassium  bichromate 10  parts 

Hot  water 50  parts 

Sulphuric  acid 50  parts 

Add  the  acid  very  cautiously  after  the  bichromate  solution  cools.  When 
the  slides  are  freed  from  balsam,  wash  them  in  water,  rinse  in  a  dilute 
solution  of  caustic  soda,  again  in  water,  and  finally  place  them  in 
ether-alcohol  until  needed. 

15.  If  Sections  Appear  Milky  or  Hazy  under  a  medium  power  of 
the  microscope,  when  finally  mounted,  the  effect  is  probably  due  to  one 
of  the  following  causes:  (a)  The  clearer  is  poor  and  needs  replenishing 
or  correcting.    (6)  The  absolute  alcohol  contains  water  (see  7).    (c)  The 


58  Animal  Micrology 

cover  bore  moisture.  Passing  a  cover-glass  quickly  through  a  flame 
before  putting  it  onto  the  object  will  remove  moisture,  (d)  The  acid  has 
not  been  entirely  removed  from  the  sections,  (e)  Too  much  albumen 
fixative  has  been  used.  (/)  The  glycerin  of  the  albumen  fixative  has  not 
been  removed  by  passing  sections  of  objects  stained  in  bulk 
(see  vi,  4)  back  into  absolute  alcohol  after  removing  paraffin 
from  them. 

The  defect  may  be  remedied  frequently  by  dissolving  off 
the  cover  in  xylol  or  turpentine,  descending  through  the 
series  of  reagents  to  the  point  where  the  fault  lies,  correcting 
and  ascending  again  according  to  the  regular  method.  To 
remove  water,  for  example,  it  is  only  necessary  to  go  back 
as  far  as  absolute  alcohol  which  has  a  great  affinity  for  water. 
16.  Dry  or  Dull-Looking  Areas  under  the  Cover-Glass  indicate 
that  the  sections  were  allowed  to  get  dry  after  the  removal 
from  the  clearer,  or  that  insufficient  balsam  was  applied. 

17.  Balsam  Which  Exudes  from  under  the  Cover  may  be  scraped  off  with 
an  old  knife  after  it  hardens.  Remove  the  last  traces  by  means  of  a 
brush  or  a  cloth  dipped  in  turpentine  or  xylol.  Balsam  may  be  removed 
from  the  surface  of  a  cover  by  means  of  a  brush  dipped  in  xylol. 

18.  If  Sections  Wash  off  the  Slide  the  defect  is  probably  due  to  one 
of  the  following  causes:  (a)  The  slide  was  soiled  or  oily.  Remedy  by 
cleaning  slides  thoroughly  (see  14).  (b)  The  albumen  fixative  is  too  old. 
(c)  The  transitions  in  the  alcohol  have  been  too  great.  This  is  true  some- 
times of  thick  sections. 

19.  Flooding  Sections  with  the  Dye  by  means  of  a  pipette,  especially 
in  case  of  stains  which  act  rapidly  (e.  g.,  eosin,  acid  fuchsin,  Lyons 
blue,  picric  acid,  etc.),  is  sometimes  more  convenient  than  immersing 
the  sections  in  a  jar  of  the  staining  fluid.  Small  bottles  with  com- 
bination rubber  stopper  and  pipette  (Fig.  31)  are  now  provided  for  this 
purpose  by  dealers. 

20.  Balsam  Mounts  in  Which  the  Stain  Has  Faded  may  frequently  be 
restained,  either  with  the  original  or  with  other  stains.  All  that  is 
necessary  is  to  dissolve  off  the  cover  in  xylol  (2  to  3  days)  and  pass  the 
preparation  down  through  the  alcohols  to  the  stain  in  the  usual  manner. 

21.  Ink  for  Writing  on  Glass  (Hubbert,  Journal  of  Applied  Micros- 
copy, Vol.  V,  p.  1680). — Mix  drop  by  drop  3  parts  of  a  13  per  cent,  alco- 
holic solution  of  shellac  with  5  parts  of  a  13  per  cent,  aqueous  solution  of 
borax.  If  a  precipitate  forms,  heat  the  solution  until  it  clears.  Add 
enough  methylen  blue  to  color  the  mass  deep  blue. 


CHAPTER  VII 

THE  CELLOIDIN  METHOD 

Use  the  tissues  which  were  prepared  for  this  method  including 
pieces  of  the  brain  and  spinal  cord  which  were  fixed  in  Erlicki's 
fluid.  Reserve  a  piece  of  spleen  for  the  freezing  method, 
chap.  viii. 

1.  Fixing,  washing,  and  dehydrating  are  the  same  as  usual 
(chap,  iii) .  If  the  object  is  in  70  per  cent,  alcohol,  complete  the 
dehydration  by  using  successively  95  per  cent,  and  absolute  alco- 
hol. It  should  remain  in  the  absolute  alcohol  for  from  12  to  24 
hours. 

2.  From  absolute  alcohol  transfer  the  object  to  equal  parts  of 
absolute  alcohol  and  ether  12  to  24  hours. 

3.  Next,  to  thin  celloidin  for  from  36  hours  to  several  days. 

4.  Thence  to  thick  celloidin  for  from  24  hours  to  several  days. 

5.  Prepare  a  wooden  block  in  such  a  manner  that  it  will  have 
surface  enough  to  accommodate  the  object,  leaving  a  small  margin, 
and  length  enough  to  be  readily  clamped  into  the  carrier  on  the 
microtome  (Fig.  32).  Dip  the  end  of  the  block  to  which  the 
object  is  to  be  attached  into  ether-alcohol  for  a  minute  and  then 
into  thick  celloidin.  Let  it  dry  so  that  later  air  bubbles  will  not 
work  up  out  of  the  wood  into  the  imbedding  mass. 

6.  Oil  one  side  of  a  strip  of  stiff  paper  by  rubbing  on  a  very 
little  vaselin,  and  wrap  it,  oiled  surface  in,  about  the  prepared 
end  of  the  block  in  such  a  way  that  it  will  project  beyond  the 
end  of  the  block,  forming  a  collar  high  enough  to  extend  a  little 
beyond  the  object  which  is  to  be  placed  within  it.  Tie  the  paper 
in  place  by  means  of  a  thread. 

7.  Pour  a  small  amount  of  thick  celloidin  into  the  paper  cup 
thus  formed  and  with  forceps  remove  the  piece  of  tissue  and  place 
it  in  celloidin.  Add  more  thick  celloidin  until  the  cup  is  full. 
By  means  of  needles  which  have  been  moistened  in  ether-alcohol 
arrange  the  object  so  that  it  will  be  cut  in  the  desired  plane. 

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Animal  Micrology 


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Chapter  VII:    The  Celloidin  Method  61 

8.  Into  a  small  stender  dish  put  chloroform  to  the  depth  of 
3  mm.  When  a  film  has  formed  over  the  exposed  surface  of  the 
celloidin  place  it  in  the  chloroform  to  harden.  It  need  not  be 
submerged.  Keep  the  vessel  tightly  covered.  The  object  may 
be  left  for  a  day  or  two,  but  1  to  3  hours  usually  suffices. 

9.  Transfer  the  block  to  70-83  per  cent,  alcohol,  where  it 
may  remain  indefinitely. 

10.  Make  a  careful  study  of  the  microtome  used  for  cutting 
celloidin  (Fig.  32). 

11.  Place  the  block  in  the  object  carrier  of  the  microtome  at 
the  proper  level  and  arrange  the  microtome  knife  obliquely,  so 
that  it  will  slice  through  the  object  with  a  long  drawing  cut  for 
at  least  half  the  length  of  the  blade.  If  the  object  is  oblong  it  is 
advantageous  to  have  the  long  diameter  parallel  to  the  edge  of 
the  knife. 

12.  Keep  both  the  knife  and  the  object  flooded  with  70  per 
cent,  alcohol. 

13.  Draw  the  knife  through  the  object  with  a  straight  steady 
pull;  avoid  pulling  down  on  or  lifting  the  knife  carrier. 

14.  If  the  feed  is  not  automatic  push  the  knife  back  to  position 
always  before  turning  the  screw  which  raises  the  object.  Cut 
the  sections  about  15  or  20  microns  thick. 

15.  As  the  sections  are  cut,  transfer  them  by  means  of  a  small 
soft  brush  or  a  paper  spatula  to  a  flat  stender  or  a  watch-glass 
containing  70  per  cent,  alcohol. 

16.  Transfer  some  of  the  sections  through  50  and  35  per  cent, 
alcohol,  2  minutes  each,  into  borax-carmine  for  from  20  to  30 
minutes,  or  until  stained  (12  to  24  hours). 

17.  Wash  successively  in  35,  50,  and  70  per  cent,  alcohols, 
leaving  the  sections  from  2  to  3  minutes  in  each. 

18.  Transfer  the  sections  to  95  per  cent,  alcohol  for  3  to  5 
minutes.  Absolute  alcohol  is  not  to  be  used  with  celloidin 
because  it  dissolves  the  celloidin. 

19.  Clear  in  carbol-xylol  for  from  10  to  20  minutes. 

20.  Mount  in  balsam  (see  chap,  vi,  I,  step  9). 


62  Animal  Micrology 

STAINING  CELLOIDIN  SECTIONS  IN  HEMATOXYLIN  AND  EOSIN 

The  objects  are  killed,  fixed,  and  preserved  as  usual  in  70  per 
cent,  alcohol,  and  sectioned  as  in  the  above  method. 

1.  Fifty  and  35  per  cent,  alcohol  each  3  to  5  minutes. 

2.  Delafield's  hematoxylin,  10  to  30  minutes. 

3.  Water  5  minutes. 

4.  Thirty-five,  50,  and  70  per  cent,  alcohol  each  3  to  5  minutes. 

5.  Acid  alcohol  until  the  celloidin  which  surrounds  the  object 
shows  but  little  of  the  stain. 

6.  Seventy  per  cent,  alcohol,  barely  alkaline  (see  chap,  vi, 
memorandum  10),  until  the  red  color  caused  by  the  acid  is  replaced 
by  bluish  purple. 

7.  Alcoholic  eosin,  30  seconds  to  1  minute. 

8.  Ninety-five  per  cent,  alcohol,  2  to  5  minutes.  Clear  in  car- 
bol-xylol  and  mount  in  balsam. 

Note. — As  time  permits  section  other  tissues  by  the  celloidin  method 
and  stain  as  above. 

MEMORANDA 

1.  If  Chloroform  is  Not  at  Hand,  80  per  cent,  alcohol  will  harden  the 
celloidin,  although  more  slowly. 

2.  The  Length  of  Time  that  objects  should  be  left  in  ether-alcohol 
and  the  celloidin  mixtures  depends  upon  the  size  and  density  of  the 
objects.  When  time  permits  it  is  always  best  to  leave  them  several  days, 
or  even  weeks  in  the  mixtures  of  celloidin.  For  large  objects  such  as  the 
medulla  of  a  large  brain  this  is  a  necessity.  For  an  embryo  of  large  size 
months  may  be  required. 

3.  Blocks  for  Celloidin  Mounting  may  be  of  wnite  pine,  glass,  vulcan- 
ized fiber  or  even  a  very  hard  paraffin.  Cork  should  not  be  used 
because  it  is  liable  to  give  or  bend.  The  vulcanized  fiber  is  the  most 
satisfactory.  It  may  be  purchased  from  dealers  in  the  form  of  strips 
which  may  easily  be  sawn  to  the  necessary  dimension.  It  is  well  to  saw 
several  parallel  cuts  into  the  upper  edge  of  the  block  to  provide  points 
of  attachment  for  the  celloidin. 

4.  Other  Clearers  may  be  substituted  for  carbol-xylol.  One  which 
clears  from  95  per  cent,  and  which  does  not  dissolve  celloidin  must  be 
chosen.  Cedar  oil  is  an  excellent  clearer  as  is  also  beech  wood  creasote. 
Other  good  clearers  are  (1)  origanum  oil,  (2)  a  mixture  of  oil  of  thyme 
(3  parts)  and  castor  oil  (1  part),  and  (3)  Eycleshymer's  clearing  fluid 


Chapter  VII:    The  Celloidin  Method  63 

which  is  a  mixture  of  equal  parts  of  bergamot  oil,  cedar  oil,  and  anhy- 
drous carbolic  acid. 

5.  Imbedding  a  Number  of  Objects  in  one  mass  is  frequently  conven- 
ient. Fold  a  stiff  paper  into  a  box  of  the  proper  size  (chap,  v,  step  6) 
or  use  metal  Ls  (Fig.  30).  Pour  in  thick  celloidin,  put  the  objects  in 
place  and  orient  them  properly  for  cutting.  Leave  a  space  of  about 
8  mm.  between  adjacent  objects.  Fill  the  box  with  thick  celloidin  and 
set  it  in  a  dish  containing  a  little  chloroform,  or  leave  it  in  80  per  cent, 
alcohol  to  harden.  When  ready  to  proceed,  cut  the  large  blocks  into 
smaller  ones  each  containing  a  piece  of  tissue.  To  fasten  it  to  the  wood, 
trim  the  small  celloidin  block  to  the  proper  dimensions,  soften  for  a  few 
minutes  in  ether-alcohol,  the  side  to  be  attached,  then  dip  it  into  thick 
celloidin  and  apply  to  the  end  of  a  wooden  block  which  likewise  has  been 
dipped  into  the  ether-alcohol  and  the  thick  celloidin.  Press  the  two 
together  and  place  them  in  chloroform  or  80  per  cent,  alcohol  to  harden. 

6.  Anilin  Dyes  are  usually  avoided  in  the  celloidin  method  because 
they  stain  the  celloidin  intensely  and  are  not  removed  in  subsequent 
treatment.  When  necessary,  however,  some  (e.  g.,  eosin)  may  be  used. 
Saffranin,  for  example,  may  be  removed  satisfactorily  from  the  celloidin 
by  means  of  acid  alcohol  without  extracting  all  the  stain  from  the  tissue. 
If  anilin  dyes  have  been  used  it  is  sometimes  better  to  remove  the  celloi- 
din by  treating  the  sections  with  absolute  alcohol  or  with  ether  before 
the  final  clearing  and  mounting. 

7.  Relative  Merits  of  the  Paraffin  and  the  Celloidin  Methods. — Celloidin  is 
good  for  large  objects,  for  brittle  or  friable  objects,  and  for  delicate  objects 
which  heat  would  injure.  It  does  not  require  removal  from  the  tissues 
ordinarily,  hence  it  holds  delicate  structures  together  permanently. 
Very  thin  sections  cannot  be  cut,  consequently  it  is  of  little  value  in 
cytological  work.  It  is  usually  impractical  to  attempt  to  cut  sections 
under  10  microns  in  thickness.  The  method,  moreover,  is  extremely 
slow.  The  paraffin  method  is  comparatively  rapid,  serial  sections  may 
be  cut  and  mounted  with  ease,  and  very  thin  sections  may  be  obtained. 
Large  objects  do  not  section  satisfactorily,  although  up  to  10  mm.  or 
even  greater  diameter  they  cut  readily.  The  rule  is  to  use  the  paraffin 
method  when  you  can. 

8.  For  Brittle  Objects,  a  Combination  of  Celloidin  and  Paraffin  Infiltration 
sometimes  proves  successful.  The  method  is  too  tedious  for  ordinary 
use  although  it  must  sometimes  be  resorted  to  with  friable  or  delicate 
objects  such  as  eggs.  Infiltrate  with  celloidin  in  the  usual  manner 
and  imbed  in  a  paper  box,  but  do  not  mount  on  a  block.  Harden 
in  80  per  cent,  alcohol,  transfer  to  95  per  cent,  alcohol  for  12  hours, 
immerse  in  pure  origanum  oil  and  95  per  cent,  alcohol  equal  parts  for  12 
hours,  then  in  pure  origanum  oil  for  2  to  3  hours;  next  transfer  to  a  mix- 


64  Animal  Micrology 

ture  of  equal  parts  of  origanum  oil  and  xylol  for  a  few  hours,  and  finally 
to  pure  xylol.  Proceed  from  this  point  as  in  ordinary  paraffin  infiltration 
and  sectioning,  although  the  length  of  time  in  the  paraffin  bath  should 
be  curtailed  as  much  as  possible  to  avoid  making  the  celloidin  brittle. 

9.  To  Transfer  Celloidin  Sections  from  the  Knife  it  is  an  excellent  plan 
to  use  a  paper  spatula;  a  bit  of  postal  card  held  in  the  cleft  end  of 
a  small  stick  answers  very  well.  Press  the  paper  down  evenly  on  the 
section  and  then  slide  it  off  the  edge  of  the  knife.  The  section  adheres 
to  the  paper.  In  carrying  loose  sections  from  one  fluid  to  another  an 
ordinary  section  lifter  may  be  used  or  a  glass  rod  around  which  the  sec- 
tion is  allowed  to  curl  answers  very  well. 

10.  Objects  Stained  in  Bulk  May  Be  Cleared  while  Yet  in  the  Block,  then 
sectioned,  and  mounted  without  passing  back  into  the  alcohols.  After 
the  block  of  celloidin  has  hardened  sufficiently  in  chloroform  it  is  trans- 
ferred directly  to  the  clearer  (cedar  oil,  or  a  mixture  of  oil  of  thyme 
3  parts  and  castor  oil  1  part).  In  cutting  objects  thus  cleared  the  knife 
must  be  flooded  with  the  clearer  instead  of  alcohol.  Do  not  allow  the 
sections  to  become  dry.  If  it  is  desired  to  use  this  method  for  a  celloidin 
block  which  has  already  been  preserved  in  70  to  83  per  cent,  alcohol,  the 
block  must  pass  through  95  per  cent,  alcohol  (1  to  2  hours)  before  it  is 
placed  in  the  clearer. 

11.  Collodion  instead  of  Celloidin  is  used  by  some  workers.  Celloidin, 
in  fact,  is  only  a  patent  preparation  of  collodion,  which  is  a  solution  of 
gun  cotton  in  ether  and  strong  alcohol.  Thin  and  thick  solutions  are 
employed  and  the  method  is  in  every  respect  similar  to  the  celloidin 
method.     Collodion  is  cheaper  than  celloidin. 

12.  Fixing  Serial  Celloidin  Sections  to  the  Slide  is  accomplished,  (1)  by 
covering  the  sections,  when  mounted  in  proper  order,  with  a  strip  of 
tissue  paper  which  is  then  bound  fast  by  wrapping  thread  around  it. 
Lee  (Microtomist's  Vade-Mecum,  6th  ed.,  p.  144)  recommends  (2)  the 
albumen  method  for  celloidin  sections  as  well  as  for  paraffin.  (3)  If  the 
sections  on  the  slide  are  carefully  flooded  with  95  per  cent,  alcohol  two 
or  three  times,  this  drained  off  and  followed  by  a  small  amount  of  ether- 
alcohol  or  ether  fumes  until  the  edges  of  the  sections  begin  to  soften  per- 
ceptibly (10  to  20  seconds),  the  sections  will  generally  adhere  to  the  slide 
sufficiently  when  the  celloidin  becomes  hard  again  upon  exposure  to  the 
air  (30  seconds)  after  the  ether-alcohol  has  been  drained  off;  they  must 
then  be  immersed  in  95  per  cent,  alcohol  before  any  further  steps  are 
taken. 

13.  Gilson's  Rapid  Celloidin  Process  (Lee,  The  Microtomist's  Vade- 
Mecum,  6th  ed.,  p.  131)  is  a  very  valuable  one  because  of  the  great  sav- 
ing of  time.  After  dehydration  the  object  is  saturated  with  ether  and 
finally  placed  into  a  test-tube  containing  thin  celloidin.     The  lower  end 


Chapter  VII:    The  Celloidin  Method  65 

of  the  tube  is  then  dipped  into  melted  paraffin  and  allowed  to  remain 
there  until  the  celloidin  solution  has  boiiod  down  to  about  one-third  of 
its  original  volume.  The  mass  is  then  mounted  in  the  ordinary  way, 
hardened  for  an  hour  or  more  in  chloroform,  and  cleared  in  cedar  oil. 
Sections  are  cut  as  directed  under  memorandum  10. 


CHAPTER  VIII 
THE  FREEZING  METHOD 

1.  Use  a  piece  of  spleen  which  has  been  properly  fixed  and 
later  preserved  in  70  per  cent,  alcohol.  Transfer  it  through  50 
and  35  per  cent,  alcohol  successively  to  water,  and  wash  it  for  12 
hours  in  running  water. 

2.  Place  it  into  a  gum  and  syrup  mass  for  24  hours  (a  saturated 
solution  of  loaf  sugar  in  30  c.c.  of  distilled  water,  added  to  50  c.c. 
of  gum  mucilage.  Prepare  a  supply  of  gum  mucilage  by  dissolving 
60  grams  of  best  gum  acacia  in  90  c.c.  of  distilled  water). 

3.  Examine  the  freezing  microtome  carefully  (Fig.  33). 

4.  Remove  the  gum  and  syrup  mixture  from  the  outside  of 
the  tissue  with  a  cloth,  put  a  little  gum  mucilage  (not  gum  and 
syrup)  on  the  freezing  disk  of  the  microtome,  and  place  the  tissue 
in  it  in  such  a  way  that  longitudinal  sections  through  the  hilum 
may  be  cut.  Surround  the  object  with  gum  mucilage  and  set  the 
freezing  apparatus  to  going.  If  carbon  dioxide  is  used,  open  the 
valve  very  cautiously,  and  let  only  a  small  quantity  of  the  gas 
escape. 

Note. — Carbon  dioxide  is  commonly  used  for  charging  soda  water 
and  beer.  It  may  be  purchased  in  iron  cylinders  containing  about  20 
pounds  of  the  liquified  gas.  The  cylinder,  when  empty,  is  exchanged 
for  a  charged  one,  so  that  the  purchaser  pays  only  for  the  contents.  The 
Bardeen  microtome  (Fig.  33)  may  be  screwed  directly  upon  the  carbon- 
dioxide  cylinder  when  the  latter  is  in  a  horizontal  position,  or,  if  desired, 
the  cylinder  may  be  placed  vertically  and  the  microtome  attached  by 
means  of  an  L-shaped  piece  of  heavy  tubing.  This  microtome  has  the 
advantage  over  the  common  forms  of  freezing  microtomes  of  wasting  less 
gas  and  of  greater  freedom  from  clogging.  It  is  advantageous  to  have 
an  extra  long  handle  to  the  key  which  is  used  for  opening  the  escape 
valve  of  the  carbon-dioxide  cylinder. 

5.  As  soon  as  the  gum  is  frozen,  continue  to  add  more  until  the 
tissue  is  completely  covered  and  frozen. 

6.  Work  the  microtome  screw  with  one  hand  and  plane  off 
sections   (15  to  20  microns  thick)  with   the   other.      The  well- 

67 


68 


Animal  Micrology 


sharpened  blade  of  a  carpenter's  plane  is  the  best  instrument  for 
cutting.     It  must  be  frequently  stropped. 

The  blade  should  be  mounted  in  a  short,  broad  handle,  which  may  be 
grasped  easily  and  firmly  with  one  hand.  In  cutting,  the  bevel  edge  of 
the  knife  should  set  squarely  on  the  glass  ways  of  the  microtome  so  that 
the  handle  of  the  knife  is  inclined  toward  the  operator  at  about  an  angle 
of  45  degrees  from  the  perpendicular.  The  hand  guiding  the  knife  should 
be  firmly  supported  against  the  chest  while  pressing  the  cutting  edge 
steadily  against  the  glass  ways  of  the 
microtome.  The  cutting  stroke  is 
made  by  bending  the  body  forward 
from  the  waist  and  thus  forcing  the 
blade  squarely  across  the  surface  of 
the  tissue. 

The  blade  must  be  kept  cold  to 
prevent  sections  from  sticking  to 
it.  If  the  sections  fly  off  or  roll, 
the  tissue  is  probably  frozen  too 
hard.  The  same  defect  may  arise 
if  there  is  insufficient  syrup  in  the 
gum  with  which  the  tissue  has 
been  saturated.  To  correct,  let  the 
tissue  thaw  a  little,  and  if  still  at 
fault,  soak  it  again  in  a  mixture 
which  contains  a  greater  propor- 
tion of  syrup.  Work  rapidly,  so 
as  to  cut  sections  in  quick  succes- 
sion. Several  sections  may  be 
allowed  to  collect  on  the  blade 
before  they  need  be  removed. 

7.  Transfer  the  sections  to  dis- 
tilled water.  The  water  should  be  changed  several  times  to  dis- 
solve out  the  gum.  Reserve  a  few  sections  in  water  for  later  use 
(step  11). 

8.  Immerse  a  few  of  the  sections  for  10  to  30  minutes  in 
Delafield's  hematoxylin,  then  wash  them  in  several  changes  of 
tap  water. 


Fig.  33.— Bardeen  Carbon-Dioxide  Freez- 
ing Microtome. 

The  freezing  chamber  contains  a 
spiral  passage  through  which  the  expand- 
ing carbon-dioxide  passes,  securing  the 
maximum    freezing    power.      The    Knife 


slides  on  glass  guides, 
twenty  microns. 


The  finest  feed  is 


Chapter   VIII:    The  Freezing  Method 


69 


9.  Transfer    the   sections  through   the    successive    grades   of» 
alcohol  (decolorizing  with  acid  alcohol  if  necessary)  up  to  absolute 
alcohol,  leaving  them  two  minutes  in  each,  after  which  remove 
them  to  carbol-xylol  for  5  minutes  or  until  clear.     If   desired, 
stain  with  eosin  (30  to  60  seconds)  after  70  per  cent,  alcohol. 

10.  Remove  one  or  two  of  the  best  to  a  slide,  drain  off  the 
excess  of  carbol-xylol,  add  a  few  drops  of  balsam  and  a  cover-glass 
of  suitable  size,  and  label. 

11.  Remove  the  sections  reserved  in  step  7  to  a  test-tube  con- 
taining a  small  amount  of  water,  and  shake  the  test- tube  vigorously 


Fig.  34.— Ether  or  Rhigolene  Freezing  Attachment. 

for  a  minute  or  two.  This  removes  the  lymphocytes  from  the 
sections,  and  exposes  the  reticular  connective  tissue  so  that  it  may 
be  examined.     Dehydrate  the  sections  and  mount  in  balsam. 

MEMORANDA 

1.  Fresh  Tissues  Are  Frequently  Sectioned  by  the  freezing  method.  The 
tissue  may  be  transferred  directly  to  the  disk  of  a  microtome  without 
previous  imbedding,  and  sectioned  after  freezing.     This  affords  a  ready 


70  Animal  Micrology 

means  of  rapidly  determining  the  nature  of  a  given  tissue,  and  is  very 
serviceable,  especially  to  the  pathologist.  The  principal  objection  is 
that  crystals  of  ice  form  in  the  cells  and  distort  them  badly.  This  is 
avoided  when  syrup  and  gum  is  used  for  imbedding. 

2.  Sections  May  Be  Preserved  in  alcohol  in  the  usual  way  after  being 
cut  by  the  freezing  method.  All  trace  of  gum  should  be  washed  out  and 
the  sections  passed  through  the  grades  of  alcohol  to  83  per  cent.,  where 
they  may  remain  indefinitely. 

3.  Sections  of  Fresh  Tissue  May  Be  Fixed  and  washed  out  after  cutting 
if  desired.  This  requires  but  little  time,  and  the  sections  will  take 
stain  much  more  satisfactorily  after  having  been  subjected  to  a  fixing 
reagent. 

4.  Objects  Which  Alcohol  Would  Injure  may  be  sectioned  by  the  freezing 
method  and  mounted  in  aqueous  media. 

5.  Ether  or  Rhigolene .  is  Sometimes  Used  for  Freezing,  although  the 
method  is  more  expensive  and  less  satisfactory  on  the  whole  than  the 
carbon  dioxide  method.  Fig.  34  shows  a  common  form  of  freezing 
attachment  used  for  either  of  these  liquids. 


CHAPTER  IX 

METALLIC  SUBSTANCES  FOR  COLOR  DIFFERENTIATION 

I.     A  GOLGI  METHOD  FOR  NERVE  CELLS  AND  THEIR  RAMIFICATIONS 

The  Golgi  chrom-silver  method  is  one  widely  used  for  the 
demonstration  of  nerve  cells  together  with  their  various  processes. 
There  are  many  modifications  of  the  method  all  of  which  are  more 
or  less  inconstant  in  their  results.  In  a  successful  preparation, 
the  various  cells  and  nerve  processes  are  not  equally  blackened,  a 
fact  which  allows  of  discrimination  between  the  different  elements. 
Sometimes  the  ganglion  cells  and  fibers  remain  unstained  while 
the  neuroglia  cells  are  impregnated,  or  occasionally  other  elements 
than  nervous  tissue  (e.  g.,  blood-vessels)  are  affected. 

The  following  method  is  applied  to  material  preserved  in  10 
per  cent,  formalin  and  is  a  so-called  "rapid  method." 

1.  From  the  brain  and  spinal  cord  which  have  previously  (see 
note,  p.  38)  been  subdivided  and  placed  in  at  least  10  times  their 
volume  of  10  per  cent,  formalin  (3  days  to  an  indefinite  time), 
cut  out  small  pieces  4  to  5  mm.  thick  from  the  region  desired  for 
study  and  transfer  them  to  a  vessel  containing  from  15  to  20  times 
their  volume  of  a  3.5  per  cent,  aqueous  solution  of  potassium 
bichromate.  They  should  remain  in  this  solution  for  from  2  to  5 
days.  Renew  the  fluid  at  the  end  of  12  hours.  Keep  the  different 
pieces  of  tissue  in  separate  vessels  so  as  to  avoid  confusion. 

2.  For  impregnation,  transfer  the  tissues  to  a  silver  nitrate 

solution  made  as  follows: 

Silver  nitrate  (crystals) 1.5  grams 

Distilled  water 200.0  c.c. 

Concentrated  formic  acid 1.0  drop 

3.  Rock  the  tissues  gently  in  a  small  amount  of  this  fluid  until 
the  brown  precipitate  of  silver  chromate  ceases  to  appear,  then 
transfer  them  into  from  20  to  40  times  their  bulk  of  fresh  silver 
nitrate  solution  and  leave  them  in  the  dark  for  from  3  to  6  days. 
Change  the  fluid  after  the  first  12  hours. 

71 


72  Animal  Micrology 

4.  Transfer  a  few  of  the  brown  pieces  of  tissue  to  95  per  cent, 
alcohol  for  half  an  hour,  renewing  it  once  or  twice  during  this 
time.  Leave  the  rest  of  the  tissue  in  the  silver-nitrate  solution 
for  future  use  in  case  the  first  attempt  proves  unsuccessful. 

5.  Remove  the  pieces  from  95  per  cent,  to  absolute  alcohol  for 
20  minutes,  changing  the  latter  once.  Then  transfer  them  to 
ether-alcohol  for  20  minutes. 

6.  Imbed  in  celloidin  without  waiting  for  infiltration  to  occur 
(thin  celloidin  30  minutes,  thick  celloidin  10  minutes).  Mount 
directly  on  a  block  and  harden  in  chloroform  for  20  minutes. 

7.  From  chloroform  transfer  directly  to  the  clearing  fluid 
(e.  g.,  cedar  oil),  and  as  soon  as  clear  (30  to  60  minutes)  cut  sec- 
tions 50  to  100  microns  thick,  only  keep  the  knife  flooded  with 
the  clearing  fluid  instead  of  alcohol.  Cut  sections  of  cortex  so 
that  they  will  be  perpendicular  to  the  surface  of  the  brain. 

8.  When  the  sections  are  thoroughly  cleared,  transfer  them  to 
a  slide  flooded  with  the  clearing  fluid,  select  such  as  prove  desir- 
able upon  microscopic  inspection,  and  discard  the  remainder. 

9.  Replace  the  oil  with  xylol,  then  remove  the  xylol  by  press- 
ing upon  the  sections  with  blotting  paper.  Add  enough  thick 
Canada  balsam  to  cover  the  sections. 

Caution. — Do  not  put  on  a  cover-glass;  moisture  must  evapo- 
rate from  the  section.  If  this  is  prevented  the  metal  deposits 
break  up  and  the  sections  become  worthless. 

10.  Keep  the  preparations  level  and  put  them  away  in  a  dry 
place  free  from  dust.  If  the  balsam  runs  off  the  sections  more 
balsam  must  be  added  at  once.  Do  not  attempt  to  examine  under 
a  high  power  until  the  balsam  is  thoroughly  hardened. 

MEMORANDA 

1.  A  Fuller  Account  of  the  Golgi  Methods  will  be  found  in  Hardesty's 
Neurological  Technique  (pp.  55-61),  or  in  Lee's  Microtomisfs  Vade- 
Mecum  (pp.  411-27). 

2.  An  Osmium-Bichromate  Mixture  is  frequently  used  instead  of  for- 
malin for  fixing  fresh  tissues.  To  85  parts  of  a  3.5  per  cent,  solution 
of  potassium  bichromate  add  15  parts  of  a  1  per  cent,  solution  of  osmic 
acid.  Small  pieces  (4  to  6  mm.  thick)  of  fresh  tissue  are  placed  in  40 
times  their  volume  of  this  mixture  and  kept  in  the  dark  for  from  12  to  24 


Chapter  IX:  Metallic  Substances  for  Color  Differentiation   73 

hours.  This  fixing  fluid  is  then  replaced  by  a  3.5  per  cent,  solution  of 
potassium  bichromate  as  in  the  case  of  material  fixed  in  formalin  (see 
above).     From  this  point  the  method  is  identical  with  the  one  given  above. 

3.  The  Determination  of  the  Elements  That  Will  Be  Impregnated  appears 
to  depend  upon  the  length  of  time  the  tissue  is  left  in  the  3.5  per  cent, 
solution  of  potassium  bichromate.  Hardesty  gives  the  following  lengths 
of  time  for  different  structures:  neuroglia,  2  to  3  days;  cortical  cells,  3  to 
4  days;  Purkinje  cells,  spinal  cord,  peripheral  ganglion  cells,  4  to  5  days; 
nerve  fibers  of  the  spinal  cord,  5  to  7  days. 

4.  Mounting  the  Sections  upon  a  Cover-Glass  is  preferred  by  some 
workers.  The  cover-slip  is  then  fastened  over  the  opening  of  a  perfo- 
rated slide  with  the  section  downward. 

5.  For  Permanently  Mounting  Golgi  Preparations  under  a  Cover-Glass, 
Huber  recommends  the  following  method:  the  sections  are  removed 
from  xylol  to  the  slide  and  the  xylol  then  removed  by  pressing 
blotting  paper  ovor  the  sections.  A  large  drop  of  xylol  balsam  is  then 
quickly  applied  and  the  slide  is  carefully  heated  over  a  flame  from  3  to  5 
minutes.  A  large  cover-glass  is  warmed  and  put  in  place  before  the 
balsam  cools. 

II.     SILVER  NITRATE  METHOD  FOR  NERVE.    (After  Hardesty) 

1.  The  fresh  nerve,  or  better  a  spinal  nerve  root,  may  be 
obtained  from  a  frog  which  has  just  been  killed.  Without 
stretching  the  nerve,  carefully  insert  beneath  it  the  end  of  a  strip 
of  postal  card  or  similar  card  which  has  been  trimmed  to  the 
width  of  50  mm.  The  nerve  when  cut  off  at  each  side  of  the 
card  will  adhere  to  it  and  remain  straight  and  at  approximately 
normal  tension. 

2.  Clip  off  the  end  of  the  card  bearing  the  nerve  into  a  clean 
vial  which  contains  0.75  per  cent,  aqueous  solution  of  silver 
nitrate.     Place  the  vial  in  the  dark  for  from  12  to  24  hours. 

3.  Transfer  the  nerve  to  pure  glycerin  on  a  slide  and  tease  the 
fibers  apart  thoroughly  under  the  dissecting  microscope. 

4.  Add  a  cover-glass  and  expose  the  fibers  to  sunlight  until 
they  become  brown  (30  minutes). 

5.  To  make  the  preparation  permanent,  take  off  the  cover  and 
remove  the  glycerin  by  means  of  filter  paper,  add  a  few  drops  of 
warm  glycerin- jelly,  put  on  a  clean  cover-glass,  and  press  it  down. 
Wipe  away  the  exuded  jelly  and  when  the  preparation  has  cooled, 


74  Animal  Micrology 

seal  the  cover  with  gold-size,  followed  by  Bell's  cement.      (See 

chap,  xiii,  iii,  A.) 

The  preparation  should  show  the  "cross  of  Ranvier"  and  the 

"lines  of  From  man." 
i 
III.     GOLD  CHLORIDE  METHOD  FOR  NERVE  ENDINGS 

1.  Trace  some  of  the  motor  nerves  of  a  reptile  or  mammal  to 
where  they  enter  the  muscles  (intercostals  are  best)  and  clip  out 
small  pieces  of  the  muscle.  Use  material  that  has  been  preserved 
in  10  per  cent,  formalin. 

2.  Place  the  bits  of  muscle  in  10  or  12  times  their  volume  of 
a  10  per  cent,  solution  of  formic  acid  in  distilled  water  and  leave 
them  for  from  30  to  40  minutes. 

3.  Transfer  the  tissue  into  from  8  to  10  times  its  volume  of  a 
1  per  cent,  solution  of  gold  chloride  in  distilled  water  for  from 
30  to  40  minutes.  Avoid  direct  sunlight.  The  muscle  should 
become  yellow  in  color. 

4.  Remove  the  tissue  without  washing  it  to  about  25  volumes 
of  a  2  per  cent,  formic  acid  solution  and  keep  it  in  the  dark  until 
it  assumes  a  purple  color  (24  to  48  hours).  When  the  fibers 
appear  reddish  violet  in  color  the  reduction  has  gone  far  enough ; 
if  they  show  a  decidedly  bluish  tinge  the  process  has  gone  too  far. 

5.  Wash  the  tissue  in  several  changes  of  distilled  water  for  an 
hour  and  transfer  a  small  piece  to  a  slide.  Tease  the  fibers  apart 
very  carefully  under  a  dissecting  lens.  Great  care  must  be  exer- 
cised to  avoid  tearing  the  nerve  fiber  from  its  endings.  Examine 
from  time  to  time  under  a  low  power  of  the  compound  micro- 
scope, and  when  a  nerve  fiber  and  its  termination  is  found, 
carefully  separate  it  as  much  as  possible  from  the  other  fibers. 

6.  Add  glycerin-jelly  and  a  cover-glass.  Seal  in  the  ordinary 
way   (chap.  xiii). 

Note. — Tissues  may  be  dehydrated  in  the  ordinary  way  and  mounted 
in  balsam  or  imbedded  in  paraffin  or  celloidin  and  sectioned. 


CHAPTER  X 

ISOLATION  OF  HISTOLOGICAL  ELEMENTS.     MINUTE 
DISSECTIONS 

I.     ISOLATION 

A.  Dissociation  by  Means  of  Formaldehyde;  ciliated  and 
columnar  epithelium. — 1.  Kill  a  frog  and  secure  the  hinder  part  of 
the  roof  of  the  mouth  and  the  stomach.  Slit  open  the  latter. 
Place  the  objects  in  the  dissociating  fluid  (see  reagent  10,  chap, 
i)  for  a  day  or  two. 

2.  Scrape  the  roof  of  the  mouth  after  removal  from  the  fluid 
and  mount  the  ciliated  cells  thus  obtained  on  a  slide.  Similarly 
remove  some  columnar  epithelium  from  the  internal  surface  of 
the  stomach  and  mount  on  another  slide. 

3.  Add  a  cover-glass  and  examine.  If  the  cells  cling  together 
in  clumps,  separate  them  by  drumming  gently  upon  the  cover- 
glass  with  the  handle  of  a  needle. 

4.  Stain  by  placing  a  drop  of  picro-carmine  (reagent  14, 
chap,  i)  on  the  slide  just  at  the  edge  of  the  cover  and  applying  a 
bit  of  filter  paper  to  the  opposite  edge  of  the  cover.  The  filter 
paper  absorbs  the  fluid  from  under  the  cover  and  the  stain 
replaces  it. 

5.  After  15  or  20  minutes  replace  the  stain  by  glycerin  in  a 
similar  manner. 

6.  If  a  permanent  preparation  is  desired  the  cover-glass  must 
be  sealed  (see  chap,  xiii),  or,  after  staining,  the  tissue  must  be 
dehydrated  and  mounted  in  balsam  in  the  usual  manner. 

B.  Isolation  of  Muscle  Fibers  by  Maceration  and  Teasing. — 
1.  Place  small  fragments  of  voluntary  muscle,  of  the  root  of  the 
tongue,  and  of  heart  muscle  of  the  frog  into  separate  vials  con- 
taining MacCallum's  macerating  fluid  (reagent  80,  Appendix  B). 
After  2  days  pour  off  the  fluid,  fill  the  vials  about  half  full  of 
water  and  separate  the  fascicles  by  shaking  the  vial.  Further 
isolate  the  fibers  by  teasing. 

75 


76  Animal  Micrology 

Teasing. — In  teasing  the  important  thing  to  remember  is  that  the 
elements  of  the  tissue  are  to  be  separated,  not  broken  up.  Both  patience 
and  sharp  clean  needles  are  indispensable.  The  process  is  best  carried 
on  under  the  lens  of  a  dissecting  microscope,  although  it  may  be  done 
without  such  aid.  A  background  which  enables  the  tissue  to  be  seen 
distinctly  should  be  selected,  black  for  colorless  or  white  for  colored 
objects.  Black-and-white  porcelain  slabs  are  made  for  this  purpose  and 
are  very  convenient.  A  good  dissecting  microscope  has  attached  beneath 
the  stage  a  reversible  plate  one  side  of  which  is  black,  the  other  white. 
Use  a  small  piece  of  tissue  and  begin  teasing  at  one  end  of  it. 

2.  With  the  aid  of  a  dissecting  microscope  carefully  tease  out 
in  water  a  number  of  fibers.  Use  a  small  piece  and,  beginning 
at  one  end,  with  both  needles  separate  the  piece  along  its  entire 
length  into  two;  likewise  further  subdivide  these  until  the  ulti- 
mate fibers  are  isolated. 

3.  Transfer  some  of  the  fibers  through  the  alcohols  and  xylol 
and  mount  in  balsam.  Stain  others  in  picro-carmine  for  5  to  10 
minutes  and  mount  in  glycerin  as  above. 

C.  Maceration  by  Means  of  Hertwig's  Fluid  (Hydra. Testis). — 
1.  The  solution  consists  of: 

0.05  per  cent,  aqueous  solution  of  osmic  acid    .     1  part 
0.2    per  cent,  acetic  acid 1  part 

Prepare  the  ingredients  for  this  mixture  by  diluting  the  stock 
solution  ( 1  per  cent. )  in  each  case  with  distilled  water.  Make  a 
separate  0.1  per  cent,  solution  of  acetic  acid  also. 

2.  Treat  a  hydra  with  the  osmic  and  acetic  acid  mixture  for 
3  minutes  and  then  transfer  it  to  the  0.1  per  cent,  solution  of 
acetic  acid.  Wash  in  several  changes  of  this  fluid  to  remove  all 
osmic  acid  and  let  the  hydra  remain  in  the  acetic  acid  for  12  hours. 

3.  Wash  in  water,  stain  in  carmalum  (reagent  34,  Appendix 
B)  or  better  in  Acid  carmine  (reagent  37),  and  mount  in  glycer- 
in as  above.  If  the  cells  are  not  sufficiently  separated,  gently 
tap  on  the  cover  glass. 

4.  Submit  small  bits  of  the  testis  of  some  animal  to  the  same 
treatment.  Stain  with  methyl  green  (reagent  56)  or  acid  car- 
mine (reagent  37). 


Chapter  X:   Isolation  of  Histological  Elements  77 

II.  MINUTE  DISSECTIONS 

A.  Alimentary  Canal  and  Nervous  System  of  Insects. — 1.  Carefully  dissect 
out  the  alimentary  canal  of  a  cockroach  and  the  central  nervous  system 
of  a  grasshopper  with  the  aid  of  the  dissecting  microscope  or  lens.  Wash 
each  by  gently  flooding  it  with  distilled  water  from  a  pipette,  and  then 
cover  it  with  Gilson's  fluid  (reagent  15,  Appendix  B)  or  corrosive  subli- 
mate (reagent  13)  for  5  minutes. 

2.  Wash  in  several  changes  of  water  during  the  course  of  half  an 
hour  and  stain  for  20  minutes  in  borax -carmine. 

3.  Wash  in  50  per  cent,  alcohol  and  decolorize  in  70  per  cent,  acid 
alcohol  until  the  objects  become  bright  scarlet  in  color. 

4.  Wash  in  95  per  cent,  alcohol  for  5  minutes  and  then  transfer  to 
absolute  alcohol  for  5  minutes,  xylol  or  turpentine  10  minutes  and  mount 
in  balsam.     Apply  cover  and  label. 

B.  Gizzard  of  Cricket  or  Katydid.  —  Pull  off  the  head  of  a  cricket  or 
katydid.  The  gizzard  usually  remains  attached  to  the  head  part.  Cut 
it  open  lengthwise,  wash  out  the  contents  and  mount  as  above;  only, 
omit  the  staining.     The  inside  should  be  turned  uppermost. 

C.  Sting  of  Wasp  or  Bee. — 1.  Place  a  wasp  or  bee  in  water,  cover  to 
keep  out  dust  and  let  it  stand  for  two  or  three  days  until  the  smell  be- 
comes unpleasant. 

2.  Wash  in  clear  water  and  squeeze  the  abdomen  gently  until  the 
sting  protrudes.  With  forceps  pull  it  out  carefully.  The  poison  gland 
and  duct  should  come  away  with  it. 

3.  Place  the  parts  removed  on  a  slide  and  under  a  lens  draw  the 
sting  out  of  its  sheath  by  means  of  a  small  needle  which  should  be 
drawn  over  the  outer  surface  of  the  sheath  from  the  base  to  the  apex  of 
the  sting. 

4.  Stain  and  follow  out  the  same  subsequent  treatment  as  for  II,  A 
or  mount  without  staining.  It  is  advisable  to  compress  the  object  be. 
tween  two  slides  as  soon  as  the  acid  alcohol  is  washed  out.  The  slides 
should  be  tied  together  and  left  in  95  per  cent,  alcohol  several  hours. 
Then  proceed  in  the  ordinary  way. 

D.  Salivary  Gland  of  Cockroach  or  Cricket. — Let  the  animal  soak  in 
water  as  for  preparation  of  sting.  When  sufficiently  decayed  pull  off 
the  head  carefully  with  forceps.  The  esophagus,  the  salivary  glands, 
and  crop  usually  come  along  with  it.  Stain  and  mount  as  for  sting. 
For  preparation  of  fresh  salivary  gland  see  Appendix  C,  II,  "  Salivary 
gland  of  Chironomous  larva." 

E.  Mouth  Parts  of  Insect. — 1.  Place  the  head  of  a  bee  or  cockroach 
in  95  per  cent,  alcohol  for  2  or  3  hours.  Transfer  to  absolute  alcohol 
for  30  minutes,  and  then  to  cedar  oil  for  30  minutes  to  an  hour. 


78  Animal  Micrology 

2.  Remove  the  head  to  a  slide  and  in  a  drop  of  the  oil  dissect  out  the 
mouth  parts.  Transfer  them  to  a  clean  slide,  remove  the  excess  of  oil 
and  arrange  them  in  their  relative  positions  in  sufficient  balsam  to  hold 
them  in  place,  then  set  the  slide  aside  in  a  place  free  from  dust  until 
the  balsam  hardens  enough  to  keep  the  parts  from  shifting.  Make  any 
necessary  rearrangement.    Add  more  balsam  and  a  cover. 

MEMORANDA 

1.  The  Cover-Glass  May  Be  Supported  by  means  of  small  wax  feet  or 
bits  of  broken  cover-glass  when  the  tissue  is  too  bulky  to  allow  the 
cover-glass  to  fit  down  closely  to  the  slide. 

2.  A  General  Rule  for  Dissociating  Tissues  is  to  use  small  pieces  of  the 
tissue  and  not  a  very  great  amount  of  the  fluid. 

3.  For  Minute  Dissections  clove  oil  is  often  a  convenient  medium.  It 
tends  to  form  very  convex  drops,  clears  well,  and  renders  the  object 
brittle;  any  or  all  of  which  properties  may  be  useful  in  such  dissections. 

4.  The  Fixation  of  Pieces  of  Macerated  Tissue  (e.  g.,  macerated  epithe- 
lium) in  0.5  to  1  per  cent,  osmic  acid  for  an  hour  or  so,  often  proves 
advantageous. 


CHAPTEK  XI 

TOOTH,  BONE,  AND  OTHER  HARD  OBJECTS 

Sectioning  Decalcified  Tooth. — 1.  Kill  a  cat  and  remove  the 
lower  jaw  (p.  53).  With  a  fine  saw  cnt  ont  about  a  quarter  of  an 
inch  of  the  bone  bearing  a  tooth  (e.  g.,  canine),  remove  as  much 
of  the  surrounding  tissue  as  possible  and  place  the  object  in 
Erlicki's  fluid  for  several  days.  Transfer  to  nitric  acid  decalcify- 
ing fluid  (reagent  11,  chap.  i).  Use  a  relatively  large  quantity 
of  the  fluid  and  change  it  each  day  until  the  tooth  is  decalcified 
(2  to  6  days).  It  is  sufficiently  soft  to  cut  when  a  needle  can  be 
thrust  into  it  easily. 

2.  Wash  it  in  repeated  changes  of  70  per  cent,  alcohol  until 
all  trace  of  the  acid  is  removed  (about  2  days)  as  shown  by 
litmus  paper. 

3.  Transfer  the  object  through  50  and  35  per  cent,  alcohol 
successively  to  running  water  and  wash  for  24  hours. 

4.  Cut  sections  by  means  of  the  freezing  microtome  as  directed 
under  that  method  (chap.  viii).  If  a  freezing  microtome  is  not 
available  use  the  celloidin  method. 

5.  After  dissolving  out  all  of  the  gum  from  the  sections  in 
distilled  water,  immerse  them  for  half  an  hour  in  picro-carmine, 
then  remove  one  or  two  of  the  best  (through  the  center  of  the 
tooth)  to  a  slide,  drain  off  the  excess  of  stain  and  add  a  few  drops 
of  melted  glycerin-jelly.     Cover  with  a  circular  cover-glass. 

6.  When  the  jelly  has  hardened,  seal  the  cover  with  gold  size 
and  when  this  is  dry,  add  a  thin  coat  of  Bell's  cement  (see 
chap,  xiii,  ii,  A,  6). 

7.  Stain  other  sections  in  1  per  cent,  osmic  acid  for  24  hours 
and  mount  in  glycerin- jelly  as  above  or  dehydrate  and  mount  in 
balsam. 

Sectioning  Decalcified  Bone. — Saw  out  a  short  piece  from  the 
femur  of  a  cat  (p.  53).     Prepare  transverse  sections  by  decalcify- 

79 


80  Animal  Micrology 

ing  and  sectioning  in  the  same  manner  as  for  teeth.  Do  not 
destroy  the  periosteum.  Prepare  likewise  longitudinal  sections 
of  a  tarsal  bone. 

Sectioning  Bone  by  Grinding. — 1.  With  a  fine  saw  cut  a  thin 
transverse  section  of  the  femur  of  a  cat.  Let  it  macerate  in  water 
until  quite  clean,  then  dry  it  carefully. 

2.  Grind  the  disk  of  bone  between  two  hones,  keeping  the 
hones  parallel  in  order  to  avoid  wedge-shaped  sections.  The  sec- 
tion is  not  thin  enough  until  fine  print  can  readily  be  distin- 
guished through  it. 

3.  Wash  the  section  thoroughly  in  water,  transfer  it  to  abso- 
lute alcohol  for  10  minutes,  then  to  pure  ether  for  half  an  hour 

4.  After  removal  from  the  ether,  clamp  it  between  two  slides 
by  means  of  a  string  or  a  rubber  band  and  let  it  dry  thoroughly. 

5.  Place  some  xylol-balsam  in  the  center  of  a  slide  and  heat  it 
for  a  few  minutes  to  drive  off  the  xylol,  then  press  the  section  of 
bone  down  firmly  into  it  and  put  on  the  cover-glass.  The  balsam 
should  not  be  thin  enough  to  penetrate  the  tissue  of  the  bone. 

MEMORANDA 

1.  Proper  Fixing  Before  Decalcification  is  necessary  for  the  best  results. 
Miiller's  fluid  (reagent  8,  Appendix  B)  is  perhaps  the  most  common 
reagent  used  in  the  fixation  of  bones  or  teeth.  It  requires  2  to  4  weeks 
to  fix  properly. 

2.  Failure  to  Stain  Properly  is  due  ordinarily  to  insufficient  washing- 
out  of  the  acid. 

3.  Teeth  and  Other  Hard  Objects  may  be  prepared  by  grinding  in  the 
same  way  as  bone. 

4.  For  Other  Decalcifying  Fluids  than  nitric  acid,  see  Appendix  B,  v. 


CHAPTER    XII 

INJECTION  OF  BLOOD  AND  LYMPH  VESSELS 

Red  Injection  Mass. — 1.  Rub  up  4  grams  of  carmine  thor- 
oughly with  8  c.c.  of  distilled  water  in  a  mortar  and  add  ammo- 
nium hydrate  drop  by  drop  until  a  transparent  red  color  results. 

2.  Soak  50  grams  of  best  French  gelatin  in  distilled  water 
until  it  is  swollen  and  soft  (18  hours) ,  then  remove  it  to  a  porce- 
lain evaporating  dish  and  melt  it  at  a  temperature  of  about  45°  C. 

3.  While  the  gelatin  is  yet  fluid,  slowly  add  the  coloring  mat- 
ter, stirring  constantly  until  a  homogeneous  mixture  is  obtained. 

4.  Before  the  mass  cools  add  also  some  25  per  cent,  acetic  acid 
solution  drop  by  drop,  stirring  thoroughly  until  the  mass  becomes 
slightly  opaque  and  the  odor  of  ammonia  gives  place  to  a  faint 
acid  smell.  Watch  for  this  change  closely,  for  a  few  drops  too 
much  of  the  acid  will  spoil  the  entire  mass  by  precipitating  the 
carmine.  If  the  ammonia  is  not  completely  neutralized,  on  the 
other  hand,  the  coloring  matter  will  diffuse  through  the  walls  of 
the  injected  vessels  and  stain  the  surrounding  tissues.  Just 
before  using,  the  mass  should  be  heated  and  strained  through 
clean  flannel. 

With  a  large  animal  it  is  advisable  to  keep  animal  and  appa- 
ratus submerged  in  warm  normal  saline  during  the  operation  of 
injection,  but  with  a  small  animal  this  is  unnecessary  if  the 
operator  works  rapidly. 

Blue  Injection  Mass. — Prepare  a  gelatin  mass  as  directed  above.  To 
the  warm  mass  add  sufficient  quantity  of  saturated  aqueous  solution 
of  Berlin  blue  to  give  the  desired  blue  color.  If  the  blue  does  not  dis- 
solve, add  a  little  oxalic  acid  to  the  mixture.  The  blue  mass  need  not 
be  made  for  the  present  practical  exercise  unless  the  student  wishes  to 
undertake  a  double  injection  as  indicated  in  memorandum  2. 

INJECTING  WITH  A  SYRINGE;    SINGLE  INJECTION 
A  common  method  of  injection  and  one  which  proves  satisfac- 
tory in  many  instances,  is  by  means  of  a  metal  or  glass  syringe. 

81 


82 


Animal  Micrology 


Although  not  as  desirable  in  the  main  as  the  method  of  continu- 
ous air  pressure,  many  good  injections  may  be  made  by  means  of 
the  syringe.  The  apparatus  (Fig.  35)  consists  of  a  syringe  fitted 
with  a  stop-cock  in  the  nozzle,  and  a  separate  tube,  known  as  the 
cannula,  which  fits  on  to  the  end  of  the  nozzle. 
The  syringes  are  made  in  different  sizes  and 
each  is  provided  with  an  assortment  of  cannulae 
to  fit  vessels  of  different  caliber. 

1.  Provide  yourself  with  several  strong 
threads  about  four  inches  in  length  for  ligat- 
ing  blood  vessels.  Have  the  red  injection 
mass  melted  and  heated  to  about  50°  C.  Also 
have  ready  some  hot  water  to  warm  the  syringe. 

2.  Kill  a  cat  or  a  rabbit  by  means  of  chloro- 
form. In  death  from  chloroform  the  blood  ves- 
sels are  left  dilated.  Work  rapidly  so  that  the 
entire  animal  may  be  injected  while  yet  warm. 
Stretch  it  out  in  a  dissecting  pan  or  tie  it  out 
onto  a  board. 

3.  Slit  the  skin  along  the  ventral  surface  of 
the  body  to  the  middle  of  the  neck  and  reflect 
it  to  the  right  and  left  sides.     Pin  it  back  out  of  the  way. 

4.  Snip  a  small  hole  through  the  body  wall  just  posterior  to 
the  ensiform  cartilage.  Insert  the  index  finger  of  the  left  hand 
to  guide  the  scissors  and  prevent  injury  to  the  underlying  organs, 
and  cut  the  costosternal  cartilages  of  the  right  side  up  to  the  first 
rib.  In  like  manner  cut  the  cartilages  of  the  left  side  up  to  the 
first  rib. 

5.  Ligate  the  sternum  tightly  as  close  to  the  first  ribs  as 
possible  to  prevent  leakage  from  cut  blood  vessels. 

6.  Cut  off  the  apex  of  the  heart  and  expose  the  ventricles. 
The  left  ventricle  is  seen  as  a  round  opening,  the  right  as  a  slit. 

7.  With  a  sponge  wrung  out  of  warm  water,  rapidly  absorb 
the  blood  from  the  thorax. 

8.  Choose  the  largest  cannula  that  the  aorta  will  admit  and 
thrust  it  through  the  left  ventricle  into  the  aorta. 


Fig.  35:— Injecting 
Syringe. 


Chapter  XII:  Injection  of  Blood  and  Lymph  Vessels      83 

9.  With  a  pair  of  fine-pointed  forceps  (preferably  with  curved 
points)  pick  up  one  end  of  a  thread  for  ligating  and  carefully 
work  it  through  under  the  aorta  (do  not  mistake  the  vena  cava 
superior  for  the  aorta).  Tie  the  thread  around  the  aorta  over 
the  cannula,  making  a  double  or  surgeon's  knot.  Draw  it  tightly 
on  the  cannula  so  that  the  latter  will  be  held  firmly  in  place. 
Run  another  thread  through  under  the  aorta  and  have  it  in  readi- 
ness to  ligate  the  aorta  when  the  cannula  is  withdrawn. 

10.  Warm  the  syringe  by  sucking  hot  water  into  it  repeatedly, 
then  fill  it  with  warm  normal  salt  solution.  Force  out  a  little  of 
the  salt  solution  into  the  open  end  of  the  cannula,  then  connect 
cannula  and  syringe  and  force  the  warm  solution  through  the 
blood  vessels  to  cleanse  them  thoroughly  of  blood. 

11.  Empty  the  syringe  completely  and  rapidly  fill  it  and  the 
cannula  with  the  warm  injecting  fluid. 

12.  Force  out  a  little  of  the  fluid  from  the  syringe  to  expel  all 
air,  and  connect  it  carefully  with  the  cannula. 

13.  Force  the  injecting  mass  into  the  blood  vessels  by  a  slow 
steady  pressure.  Begin  with  a  very  low  pressure,  so  that  the 
large  vessels  will  be  thoroughly  filled  before  the  mass  enters  the 
capillaries.  The  pressure  should  be  gradually  increased.  Avoid 
sudden  increase  of  pressure  or  too  strong  pressure,  for  either  may 
cause  a  rupture  of  the  blood  vessels  and  consequent  extravasa- 
tion. From  8  to  10  minutes  is  about  the  time  required  to  make 
a  good  injection  of  the  cat. 

14.  Examine  the  intestines  and  the  gums  from  time  to  time 

and  also  the  inside  of  the  thigh  (from   which  the   skin  has  been 

reflected) ;  they  should  be  deeply  colored  by  the  mass  before  the 

injection  is  complete.     If  the  mass  begins  early  to  flow  from  the 

right  ventricle,  the  ventricle  should  be  ligated.     In  any  event,  it 

is  well  to  tie  the  ventricle  a  few  minutes  before  completion  of  the 

injection,  to  insure  filling  of  all  blood  vessels. 

Note. — If  the  gums  remain  uncolored,  the  cannula  has  probably 
been  forced  past  the  arteries  which  lead  to  the  head.  In  such  a  case, 
complete  the  injection  of  the  trunk  and  then,  if  injected  tissue  from  the 
head  region  is  desired,  cut  obliquely  into  one  side  of  the  innominate 
artery,  tie  a  cannula  in  place  and  inject  toward  the  head  as  in  the  case 
of  the  aorta. 


84  Animal  Micrology 

15.  When  the  injection  is  complete,  shut  the  stop-cock,  ligate 
the  aorta,  or  clamp  it  with  pressure  forceps  beyond  the  end  of 
the  cannula  and  then  remove  the  latter. 

16.  Place  the  animal  in  cold  water  or  cold  alcohol  for  half  an 
hour,  then  remove  pieces  of  liver,  spleen,  pancreas,  stomach,  intes- 
tine, salivary  glands,  kidneys,  and  voluntary  muscle  and  harden 
in  absolute  alcohol,  or  in  10  per  cent,  formalin. 

17.  When  sufficiently  hardened  transfer  the  objects  to  ether- 
alcohol  and  proceed  to  imbed  and  cut  in  celloidin  according  to  the 
method  already  given.  Make  longitudinal  sections  of  the  kidney 
parallel  to  its  flat  surface.  Cut  transverse  sections  of  liver, 
stomach  and  intestine,  longitudinal  of  the  muscle,  and  sections 
passing  longitudinally  through  the  hilum  of  the  salivary  glands 
and  spleen.  The  sections  should  not  be  under  30  microns  thick. 
Mount  some  unstained;  stain  others  in  diluted  Delafield's  hema- 
toxylin or  in  hemalum. 

MEMORANDA 

1.  Apparatus  for  Continuous  Air  Pressure  Injections  is  now  provided  in 
many  laboratories.  If  a  regular  cylinder  for  air  pressure  is  not  present, 
however,  an  assistant  with  a  little  ingenuity  can  readily  fit  up  a  suitable 
apparatus.  A  carboy  or  large-mouthed  bottle  which  can  be  tightly 
corked  will  answer  as  a  chamber  for  compressed  air,  a  water  tap,  or  a 
tank  of  water  elevated  to  the  height  of  7  or  8  feet  will  provide  sufficient 
pressure.  By  making  the  proper  connections  by  means  of  rubber  and 
glass  tubing  a  steady  stream  of  compressed  air  may  finally  be  conducted 
to  a  flask  containing  the  injection  mass;  the  flask  works  in  the  same  way 
as  an  ordinary  wash  bottle.  All  corks  and  fittings  must  be  tightly 
secured  with  wire  or  strong  cord.  If  desired,  by  adding  an  extra  per- 
foration to  the  cork  in  the  air  chamber,  a  mercury  manometer  may  be 
added  to  register  the  amount  of  air  pressure.  If  a  metal  cannula  is  not 
at  hand  a  glass  one  may  be  made  as  indicated  under  memorandum  8. 
In  lieu  of  a  stop-cock,  use  a  pinch-cock  on  the  rubber  delivery  tube. 

2.  A  Double  Injection  of  the  Vascular  System  may  be  made  by  first 
injecting  the  blue  mass  until  it  is  seen  to  flow  from  the  right  ventricle, 
then  detaching  the  tube  which  conveys  the  blue  mass,  and  slipping  over 
the  end  of  the  cannula  a  tube  conveying  a  red  mass.  This  second  mass 
should  be  in  a  bottle  or  flask  connected  with  the  pressure  bottle  by 
means  of  an  additional  tube  through  the  cork  of  the  latter,  or  the  two 
flasks  containing  the  colored  masses  may  be  connected  with  the  tube 


Chapter  XII:  Injection  of  Blood  and  Lymph  Vessels     85 

from  the  pressure  bottle  by  means  of  a  Y-tube.  Each  must  be  provided 
with  a  pinch-cock  or  clamp  to  hold  back  its  contents  while  the  other  is 
in  operation.  If  a  syringe  is  used,  it  is  better  to  have  a  second  syringe 
for  the  second  mass,  although  one  will  answer  if  it  is  rinsed  out  with  hot 
water  before  being  filled  with  the  second  mass.  The  second  mass  should 
have  a  quantity  of  very  finely  pulverized  starch  mixed  with  it,  so  that 
when  it  reaches  the  capillaries  they  will  become  completely  plugged. 

It  should  be  borne  in  mind  that  the  larger  veins  cannot  be  injected 
in  a  direction  contrary  to  their  flow,  because  of  the  valves  they  contain. 

3.  The  Lungs,  Liver  and  Kidney  are  readily  injected  through  their 
larger  blood  vessels  with  two  masses,  and  afford  very  instructive  material 
when  thus  prepared.  A  triple  injection  of  the  liver  may  be  made  by 
injecting  the  hepatic  artery  and  the  hepatic  and  portal  veins.  The  third 
mass  may  be  colored  with  China  ink.  Whitman  (Methods  in  Micro- 
scopical Anatomy  and  Embryology)  recommends  first  injecting  the 
hepatic  artery  and  afterward  the  two  veins.  The  blood  should  be  washed 
out  of  the  organ  to  be  injected  with  warm  salt  solution. 

4.  To  Inject  Lymphatics  the  puncture  method  is  commonly  employed. 
For  example,  an  aqueous  solution  of  Berlin  blue  is  drawn  into  a  hypo- 
dermic syringe,  the  sharp  point  of  the  cannula  is  thrust  into  the  tissue, 
and  the  syringe  emptied  by  slight,  steady  pressure.  For  practice,  thrust 
the  cannula  into  the  pad  of  a  cat's  foot,  and  force  in  some  of  the  injection 
mass.  If  the  leg  is  rubbed  upward,  the  fluid  will  flow  along  the  lymph 
channels  and  into  the  glands  of  the  groin. 

5.  To  Keep  Gelatin  Injection  Masses  let  them  congeal,  then  cover 
the  surface  with  95  per  cent,  alcohol,  and  leave  in  a  well-stoppered  vessel 
until  needed. 

6.  Injection  through  the  Femoral  Artery  is  frequently  practiced,  and 
is  preferred  to  injection  through  the  aorta  by  some  workers.  An 
oblique  cut  is  made  in  one  side  of  the  artery  and  the  cannula  inserted 
pointing  toward  the  heart.  Others  prefer  to  cut  into  the  dorsal  aorta 
and  inject  both  anteriorly  and  posteriorly. 

7.  The  Injecting  Syringe  must  work  without  jerking  or  catching 
along  the  wall  of  the  barrel.  It  should  always  be  carefully  cleaned  after 
using.  If  the  piston  does  not  fit  the  barrel  tightly  enough  it  should  be 
wrapped  with  gauze. 

8.  Glass  Cannulae  may  be  made  by  grasping  the  ends  of  a  short 
piece  of  soft  glass  tubing  and  heating  the  middle  in  a  flame  until  the 
glass  becomes  soft,  which  is  indicated  by  the  yellow  color  of  the  flame. 
The  tubing  should  be  constantly  rotated,  so  that  all  sides  heat  equally. 
When  the  glass  becomes  soft,  draw  the  tube  out  steadily  until  the 
diameter  of  the  soft  portion  becomes  as  small  as  desired.  When  the  glass 


86  Animal  Micrology 

has  cooled,  the  tube  should  be  cut  with  a  file  at  the  proper  place  to  make 
two  cannulae  of  it. 

9.  If  the  Blue  Color  Fades  in  the  gelatin  mass  in  the  tissues,  it  may 
frequently  be  restored  by  treating  the  tissue  or  section  with  oil  of  cloves 
or  turpentine. 

10.  A  Cold  Fluid  Gelatin  Mass  has  been  used  very  successfully  by 
Tandler  (see  abstract  by  A.  M.  C.  in  Journal  of  Applied  Microscopy, 
Vol.  V,  p.  1625).  To  prepare  the  mass,  dissolve  5  grams  of  finest  gelatin 
in  100  c.c.  of  tepid  distilled  water.  Color  to  the  desired  shade  with 
Berlin  blue,  and  then  add  slowly  5  to  6  grams  of  potassium  iodide.  The 
mass  remains  fluid  at  ordinary  temperatures,  but  when  injected  objects 
are  placed  in  5  per  cent,  formalin,  it  sets  completely  and  is  thereafter 
unaffected  by  reagents.  The  minutest  vessels  are  injected,  and  sections 
may  be  stained  in  the  usual  ways.  Subjection  to  strong  acids,  such  as 
sulphuric  or  hydrochloric,  does  not  affect  the  mass,  hence  it  may  be  used 
for  injecting  specimens  that  are  to  be  decalcified  afterward.  To  preserve 
the  fresh  mass,  add  a  few  crystals  of  thymol  and  keep  in  a  stoppered 
bottle. 

11.  Corrosion  of  Injected  Vessels  or  Cavities  is  sometimes  practiced. 
A  mass  must  be  employed  which  will  not  be  attacked  by  the  reagent 
used  for  destroying  the  surrounding  tissues.  One  of  the  best  masses 
consists  of  white  wax  5  parts  and  colophonium  6  parts,  melted  together 
at  a  temperature  of  about  75°  C.  For  fine  vessels  increase  the  propor- 
tions of  wax,  for  larger  ones  add  more  colophonium.  Vermilion,  Prussian 
blue,  or  chromate  of  lead  may  be  used  for  coloring.  The  part  to  be 
injected  should  be  placed  in  warm  water  and  the  mass  injected  at  a 
temperature  of  from  50°  to  60°  C.  The  injected  part  is  left  in  cold  water 
for  from  1  to  2  hours,  and  is  then  corroded  in  pure  hydrochloric  acid  for 
from  6  to  48  hours,  according  to  the  resistance  of  the  tissue.  Finally, 
wash  the  preparation  thoroughly  in  running  water.  For  bibliography 
and  more  detailed  directions  see  Technique  des  Injections,  par  Her- 
mann Joris,  University  Libre  de  Bruxelles,  1903. 


CHAPTER  XIII 

OBJECTS    OF    GENERAL    INTEREST:    CELL-MAKING,    FLUID 

MOUNTS,  "INTOTO"  PREPARATIONS,  DRY  MOUNTS, 

OPAQUE  MOUNTS 

When  objects  of  considerable  thickness  are  to  be  mounted  it 
is  sometimes  necessary  to  resort  to  cells  which  will  contain  the 
object  and  support  the  cover-glass.  Fluid  mounts  and  aqueous 
media  must  occasionally  be  resorted  to  for  delicate  objects  which 
would  be  injuriously  affected  by  alcohol,  or  which  are  unsuitable 
for  mounting  in  balsam.  When  such  mounts  are  used,  whether 
in  a  cell  or  not,  the  cover-glass  must  ordinarily  be  sealed  with  a 
cement  if  the  preparation  is  to  be  permanent.  In  all  cases  where 
it  is  at  all  practicable,  balsam  mounts  are  to  be  preferred  for 
permanent  preparations.  Glycerin  is  a  convenient  mounting 
medium  for  many  objects,  especially  for  temporary  mounts.  It  is 
often  used  where  such  media  as  balsam  would  render  the  prepara- 
tion too  transparent;  it  is  much  more  favorable,  moreover,  to  the 
preservation  of  color  than  are  resinous  media.  For  making  cells 
and  sealing  circular  covers,  a  turntable  (Fig.  36)  is  desirable, 
although  the  work  may  be  done  by  following  a  guide  ring  drawn 
on  paper  and  placed  under  the  slide. 

I.  TURNING  CELLS 
Prepare  12  or  15  slides  as  follows:   1.  Place  a  slide  on  a  turn- 
table and  adjust  it  so  that  its  center  lies  over  the  center  of  the 
turntable. 

2.  Dip  a  small  camel's  hair  pencil  into  gold  size,  but  do  not 
take  up  enough  of  the  fluid  to  drop. 

3.  Choose  a  guide  ring  on  the  turntable  which  is  of  slightly 
smaller  diameter  than  the  cover-glass  to  be  used,  whirl  the  table 
and  hold  the  pencil  lightly  over  the  guide  ring.  The  ring  which 
has  been  spun  should  be  even.  If  it  is  not,  practice  turning  rings 
until  satisfactory  ones  are  made.  If  the  gold  size  is  old  it  is 
probably  too  thick  to  make  suitable  rings.     Pure  linseed  oil  may 

87 


88  Animal  Micrology 

be  used  to  dilute  it,  but  it  is  advisable  to  use  only  fresh  gold  size 
if  it  is  obtainable. 

4.  The  slide  must  be  set  aside  to  dry  before  it  can  be  used  for 
mounting.     A  gentle  heat  will  aid  in  drying. 


Fig.  36— Turntable. 

5.  To  some  of  the  cells  add  successive  coats  of  gold  size  as  the 
previous  one  dries,  so  that  you  will  have  cells  of  varying  depth. 

II.  MOUNTING  IN  GLYCERIN 

A.  Water  Mites  and  Transparent  Larvae. —  1.  Kill  several 
small,  colored  water  mites  or  transparent  larvae  of  insects  by 
means  of  chloroform  (a  few  drops  in  water)  and  place  them  for 
half  an  hour  (two  or  three  hours  for  larger  objects)  into  a  mixture 
of  water  and  glycerin  equal  parts,  after  which  transfer  them  to 
pure  glycerin, 

2.  Apply  a  thin  coat  of  gold  size  to  the  upper  edge  of  a  cell 
which  is  of  sufficient  depth  to  accommodate  the  object. 

3.  Breathe  into  the  cell  to  moisten  it  so  that  the  glycerin  will 
adhere  throughout  and  prevent  the  formation  of  air-bubbles. 

4.  Fill  the  cell  flush  with  glycerin  and  put  the  object  into  it, 
carefully  spreading  out  all  parts. 

5.  Breathe  on  the  lower  surface  of  a  clean  cover-glass,  put  one 
edge  down  on  the  edge  of  the  cell  and  then  gradually  lower  the 


Chapter  XIII:    Objects  of  General  Interest  89 

cover  so  as  to  avoid  bubbles  of  air.  When  in  place,  press  the 
cover  down  gently  with  the  handle  of  a  needle  and  see  that  it 
adheres  all  around.  Wash  off  the  exuded  glycerin  and  carefully 
wipe  the  slide  with  a  cloth. 

6.  Turn  a  comparatively  broad  ring  around  the  edge  of  the 
cover  to  seal  it,  and  when  this  is  dry  add  a  very  thin  coat  of 
Bell's  cement.  Label  and  put  away  in  a  horizontal  position  until 
dry. 

Caution. — It  is  indispensable  that  the  edges  of  the  cover-glass 
be  perfectly  dry  before  attempting  to  seal  the  preparation ;  other- 
wise the  cement  will  not  adhere. 

B.  Killing  and  Mounting  Hydra. —  1.  With  a  dipping-tube 
remove  a  hydra  to  a  warm  watch-glass  and  leave  it  in  only  a  few 
drops  of  water.  Have  ready  some  hot  Gilson's  fluid  or  corrosive 
acetic,  and  when  the  hydra  sends  out  its  tentacles  and  expands  its 
body,  apply  the  reagent  by  suddenly  squirting  it  into  the  watch- 
glass  so  that  it  sweeps  over  the  hydra  from  aboral  to  oral  extremity 
and  carries  the  tentacles  out  straight.  Then  fill  the  watch-glass 
with  the  hot  fluid. 

2.  After  5  minutes  pour  off  the  fixing  fluid  and  wash  the 
animal  thoroughly  in  50  followed  by  70  per  cent,  alcohol  to  which 
a  little  tincture  of  iodine  has  been  added. 

3.  Replace  the  alcohol  with  borax-carmine  or  dilute  hema- 
toxylin and  stain  for  from  30  minutes  to  several  hours. 

4.  Remove  the  stain  with  a  pipette  and  replace  it  with  a  mix- 
ture of  equal  parts  of  glycerin  and  water  for  half  an  hour,  followed 
by  pure  glycerin.      Proceed  farther  as  in  the  preceding  exercise. 

Note. — After  removal  from  the  stain,  if  necessary,  decolorize  in  acid- 
ulated water  or  alcohol  (0.5  per  cent,  hydrochloric  acid),  then  wash  out 
the  acid  thoroughly  in  tap  water. 

Hydra  may  also  be  dehydrated,  cleared  and  mounted  in  bal- 
sam.    See  also  Appendix  D,  "Hydra." 

III.     MOUNTING  IN  GLYCERIN- JELLY 
Glycerin-jelly   is  frequently   preferable   to  pure  glycerin  for 
mounting  because  it  is  a  solid  at  ordinary  temperatures.     One 
formula  for  making  it  is  as  follows. 


90  Animal  Micrology 

Water 42  c.c. 

Gelatin 6  grams 

Glycerin .50  c.c. 

Carbolic  acid  crystals 2  grams 

Dissolve  the  gelatin  in  the  water  and  add  the  glycerin  and  the 

carbolic  acid.      Warm  for  10  or  15  minutes  stirring  continually 

until  the  mixture  is  homogeneous.     Do  not  heat  above  75°  C.  or 

the  gelatin  may  be  transformed  into  metagelatin  which  will  not 

harden  at  ordinary  temperatures.     Filter  through  fine  hot  flannel. 

Use  only  clean  gelatin  of  the  best  quality. 

A.  Small  Crustacea. —  1.  By  means  of  a  dipping- tube   isolate 

such  small  creatures  as  Cyclops,  Daphnia,  or  Cypris. 

2.  Kill  by  warming  slowly  in  a  drop  of  water  on  a  slide. 

3.  Place  them  in  a  cell  of  proper  depth,  draw  off  all  water 
with  a  pipette,  and  gently  warm  the  slide. 

4.  Place  the  bottle  of  glycerin-jelly  into  a  vessel  containing 
warm  water  until  the  jelly  becomes  liquid,  but  do  not  let  it  get 
any  warmer. 

5.  Fill  the  cell  flush  with  the  warm  jelly  and  arrange  the 
objects  in  suitable  positions. 

6.  Breathe  upon  the  lower  surface  of  a  clean  cover-glass  and 
put  it  in  place  in  the  usual  way. 

7.  Wash  away  any  trace  of  the  jelly  from  the  outside  of  the 

cell  and  when  the  slide  is  dry  run  a  ring  of  gold-size   cement 

around  the  edge  of  the  cover.     After  this  dries  varnish  with  Bell's 

cement.      It  is  not  an  absolute  necessity   to  seal   glycerin-jelly 

mounts,  but  the  writer  has  always  found  it  a  wise  precaution. 

B.  Muscle  of  Insect. — 1.  Cut  off  the  head  of  an  insect  and  bisect  the 
trunk  so  as  to  expose  the  interior.  Observe  two  kinds  of  muscular  tissue, 
that  of  grayish  color  belonging  to  the  legs,  the  yellowish  to  the  wings. 

2.  Take  a  shred  of  muscle  and  on  a  dry  slide  carefully  separate 
pieces  of  muscle  fiber  and  stretch  them  out,  while  keeping  them  moist 
by  breathing  on  them. 

3.  Mount  in  glycerin-jelly  as  directed  in  the  previous  exercise.  See 
also  Appendix  C,  IX. 

IV.     MOUNTING  IN  BALSAM 

A.  Flat  Worms. —  1.  Obtain  specimens  of  Planaria  from   the 

under  surface  of  flat  rocks  in  the  edge  of  streams  (see  Appendix 

D,  "Planaria"). 


Chapter  XIII :    Objects  of  General  Interest  91 

2.  Place  the  animal  in  a  little  tepid  water.  Watch  until  it 
is  extended  full  length,  then  flood  it  quickly  with  hot  corrosive 
sublimate,  or  hot  Gilson's  fluid.  The  animal  may  be  removed 
after  10  or  15  minutes  and  washed  thoroughly  in  50  per  cent, 
alcohol  to  which  a  little  tincture  of  iodine  has  been  added. 

3.  Stain  for  24  hours  in  borax-carmine,  or  in  Delafield's 
hematoxylin  diluted  one-half  with  water. 

4.  Wash  in  water  followed  by  35  and  50  per  cent,  alcohol 
each  15  minutes. 

5.  Decolorize  in  acid  alcohol  until  the  color  ceases  to  come 
away  freely  (10  to  30  minutes). 

6.  Wash  out  the  acid  in  70  per  cent,  alcohol,  using  the  alka- 
line alcohol  if  hematoxylin  was  used  in  staining. 

7.  Flatten  the  animal  by  compressing  it  between  two  slides 
by  means  of  a  rubber  band,  and  place  it  for  24  hours  in  95  per 
cent,  alcohol. 

8.  Transfer  to  absolute  alcohol  for  1  hour,  and  to  xylol  until 
clear. 

9.  Mount  in  balsam  in  a  thin  cell  or  without  a  cell  at  pleas- 
ure. If  on  examination  the  separate  organs  of  the  animal  are 
not  seen  distinctly,  the  reason  is  probably  that  the  object  has  not 
been  compressed  sufficiently.  This  difficulty  may  also  sometimes 
be  overcome  in  a  measure  by  letting  a  cover-glass  rest  upon  the 
live  Planarian  to  flatten  it  out  slightly,  and  then  running  the  fixing 
fluid  under*  the  cover.  Specimens  which  have  been  in  the  labo- 
ratory for  some  weeks  or  months  make  better  preparations  than 
those  fresh  from  the  stream. 

B.  Mosquito,  Gnat,  or  Aphid. —  1.  Kill  a  mosquito  with  cya- 
nide or  chloroform  and  place  it  in  cedar  oil  or  turpentine  for  an 
hour. 

2.  Remove,  and  place  it  on  its  back  on  filter  paper.  Care- 
fully spread  the  legs  of  the  insect,  put  a  drop  of  thick  balsam  on 
a  slide,  invert  the  slide,  and  bring  the  balsam  in  contact  with  the 
thorax  of  the  mosquito.  Spread  the  wings  and  the  legs  of  the 
insect  and  gently  press  it  down  into  the  balsam. 

3.  Add  thinner  balsam,  see  that  the  proboscis  and  antennae 


92  Animal  Micrology 

are  floated  out  properly,  then  add  more  balsam,  and  put  on  a 
cover-glass. 

V.     OPAQUE  MOUNTS 

Some  objects  are  mounted  to  be  viewed  by  reflected  instead  of  trans- 
mitted light.  They  may  be  mounted  in  the  ordinary  way,  and  when 
examined  as  opaque  objects,  the  light  from  the  mirror  should  be  turned 
away  and,  if  necessary,  a  strip  of  dark  paper  placed  under  the  slide  to 
shut  off  all  light  from  below. 

A.  Beetles. — Choose  a  shallow  cell  for  mounting  the  wing  cases  and 
legs  of  one  of  the  Curculionidae,  preferably  Curculio  imperalis,  the 
South  American  diamond  beetle. 

1.  Soak  the  part  in  cedar  oil  or  turpentine  for  half  an  hour,  then 
place  it  in  the  cell  in  the  proper  position,  the  outer  side  of  the  case 
toward  the  observer. 

2.  Fill  up  the  cell  with  balsam  and  add  the  cover. 

B.  Wings  of  Moths  or  Butterflies. — Prepare  parts  of  the  wings  of  moths 
or  butterflies  as  in  A.  The  wing  of  the  clothes  moth  makes  a  good 
opaque  mount.  i 

C.  Head  of  a  Fly. — 1.  Secure  the  specimen  (preferably  one  having 
colored  eyes,  as  one  of  the  gad-flies)  and  choose  a  cell  of  the  proper  size 
for  it.  The  cell  should  be  of  such  a  depth  that  the  cover  will  rest  lightly 
upon  the  object  and  retain  it  in  the  center  of  the  cell.  The  head  should 
present  the  front  view  when  mounted. 

2.  Spin  a  very  thin  coat  of  gold  size  on  to  the  dry  edge  of  the  cell  so 
that  the  cover  will  adhere. 

3.  Soak  the  head  of  the  fly  for  a  couple  of  hours  in  equal  parts  of 
glycerin  and  water. 

4.  Moisten  the  cell  by  breathing  into  it,  fill  it  with  glycerin  and 
transfer  the  object  to  it. 

5.  Breathe  on  the  cover-glass  and  apply  it  very  carefully  to  avoid 
air-bubbles.  When  the  cover  settles  into  place,  press  it  down  gently  to 
make  it  adhere  to  the  cement. 

6.  Set  it  aside  to  harden.  When  hard,  seal  on  the  turntable  with 
gold-size  followed  by  Bell's  cement  when  the  gold-size  is  dry. 

D.  Foreleg  of  Dytiscus,  the  Great  Water  Beetle. — 1.  Detach  the  foreleg 
of  a  male,  and  soak  it  in  10  per  cent,  potash  solution  (see  Appendix  B, 
reagent  76)  for  a  day  or  two. 

2.  Wash  it  in  water,  run  it  up  to  95  per  cent,  alcohol,  and  leave  it 
there  for  24  hours. 

3.  Pass  it  through  absolute  alcohol  and  clear  in  cedar  oil,  turpentine 
or  xylol. 


Chapter  XIII:    Objects  of  General  Interest  93 

4.  Lay  the  leg,  disk  side  uppermost,  in  a  drop  of  balsam  on  a  slide, 
add  another  drop  of  balsam  and  carefully  cover  with  a  clean  cover-glass. 
Place  a  small  weight  (e.  g.,  half  of  a  bullet)  on  top  of  the  cover  to  hold 
it  down  until  the  balsam  hardens. 

VI.     DRY  MOUNTS 

A.  Scales. — Prepare  a  very  shallow  cell  and  let  it  dry.  Thoroughly 
dry  the  scales  from  a  moth's  wing  by  gently  heating  them  on  a  slide 
over  a  flame.  Place  the  scales  in  a  cell,  warm  the  slide  until  the  cell 
wall  becomes  sticky,  put  on  the  cover  and  press  it  down  until  it  adheres 
all  around,  and  finally  seal  as  in  previous  exercises. 

B.  Eggs  of  Butterflies,  Small  Feathers,  Antennae  of  Insects,  etc.,  may  be 
mounted  as  dry  objects.  Care  must  be  taken  to  have  them  perfectly 
dry,  or  they  will  in  time  cloud  the  cover  with  moisture  from  within. 

MEMORANDA 

1.  Small  or  Soft  Insects  or  Their  Larvae  may  frequently  be  mounted 
directly  in  glycerin,  or  they  may  be  dehydrated  and  mounted  in  balsam. 
A  method  often  used  is  to  kill  them  in  strong  carbolic  acid  and  mount 
them  directly  in  balsam.  The  carbolic  acid  both  dehydrates  and  clears. 
It  is  better,  however,  to  clear  the  preparation  further  by  immersion  in 
cedar  oil  or  xylol  before  adding  the  balsam. 

2.  Insects  Having  Hard  Shells  must  first  be  soaked  in  10  per  cent,  pot- 
ash to  soften  them  and  render  them  transparent  if  they  are  to  be  exam- 
ined by  transmitted  light.  The  softer  parts  of  insects  so  treated  are 
destroyed  and  only  the  external  parts  remain.  Such  insects  may  be 
mounted  in  glycerin  or  glycerin-jelly,  or  they  may  be  dehydrated, 
cleared  and  mounted  in  balsam. 

3.  Delicate  Insects,  which  are  too  frail  to  withstand  much  handling, 
may  be  placed  at  once  in  cedar  oil  or  turpentine  and  after  an  hour 
mounted  in  balsam. 

4.  Wings,  Legs,  Antennae,  Mouth-Parts,  etc.,  of  Such  Forms  as  Flies  and 
Bees  which  have  been  preserved  in  alcohol,  should  be  completely  dehy- 
drated, cleared  and  mounted  in  balsam  in  cells  of  the  proper  depth. 

5.  Transparent  and  Soft  Insects  may  be  stained  in  borax-carmine  or 
hematoxylin  in  the  ordinary  way  and  mounted  as  whole  objects,  if 
desired.  They  will  stain  better  if  they  have  been  fixed  previously  in 
some  corrosive  sublimate  mixture  and  then  washed  properly  (see  Appen- 
dix B,  reagent  13).    To  stain,  follow  the  method  outlined  in  IV,  A. 

6.  To  Center  an  Object  in  a  Cell  (the  head  of  an  insect,  for  example), 
thread  a  fine  needle  with  a  hair  and  run  it  through  the  object.  Remove 
the  needle  and  imbed  the  ends  of  the  hair  in  the  cement  on  opposite 
sides  of  the  cell.    When  the  cover-glass  is  put  in  place  the  object  may  be 


94  Animal  Micrology 

adjusted  by  pulling  the  hair.  After  the  slide  is  finished  and  dry,  the 
ends  of  the  hair  should  be  cut  off  at  the  edge  of  the  cell. 

Another  method  which  will  frequently  answer  for  an  object  to  be 
mounted  in  balsam  is  to  place  the  object  (after  clearing)  in  the  center  of 
the  cell,  coat  it  with  balsam,  adjust  it  properly,  and  then  set  the  slide 
away  in  a  place  free  from  dust  till  the  balsam  thickens.  Finally  fill  the 
cell  with  balsam  and  add  the  cover. 

7.  The  Radula  or  Lingual  Ribbon  of  the  Snail  or  Slug  should  be  dissected 
out  and  soaked  for  a  day  or  two  in  a  10  per  cent,  solution  of  potash.  If 
the  animal  is  a  small  one,  cut  off  the  head  including  the  buccal  mass 
and  soak  it  in  a  solution  of  potash  until  the  soft  tissues  are  destroyed 
and  only  the  radula  remains.  From  the  potash  the  radula  is  transferred 
to  water  and  washed  for  some  hours.  With  a  strip  of  paper  on  each 
side  to  prevent  crushing  it,  it  should  be  placed  between  two  slides,  and 
the  slides  bound  together  by  means  of  string  or  rubber  bands.  While 
held  in  this  position,  dehydrate  and  clear  it.  Finally  remove  one  slide 
and  the  paper  and  mount  the  object  in  balsam  on  the  other  slide.  A 
shallow  cell  may  be  used  if  desired. 

8.  Flukes  and  Tapeworms  are  prepared  in  the  same  manner  as  planaria 
(see  IV,  A).  The  time  of  immersion  in  the  various  fluids  should  be 
lengthened  in  proportion  as  the  object  is  larger  than  the  planarian. 
See  also  Appendix  D. 

9.  Spirogyra,  Protococcus,  Volvox,  Desmids,  etc.,  may  be  mounted  in  a 
cell  in  the  following  copper  solution : 

Acetate  of  copper 1.    gram 

Camphor  water 240.    c.c. 

Glycerin 240.    c.c. 

Glacial  acetic  acid 0.3  c.c. 

Corrosi  ve  sublimate,  sat urated  aqueous  solution      0.1  c.c. 

Mix  thoroughly,  filter  and  keep  in  a  glass-stoppered  bottle.  The  green 
color  of  the  plant  may  frequently  be  preserved  for  some  time  in  this 
medium.  The  specimen  is  washed  in  water,  transferred  to  the  cell,  then 
the  solution  is  added.    The  cell  is  covered  and  sealed  in  the  usual  way. 

10.  A  Dipping-Tube  is  a  simple  glass  tube.  To  operate  it,  hold  the 
tip  of  the  forefinger  over  the  upper  end  and  dip  the  lower  end  into  the 
water  until  it  comes  just  above  the  object  desired;  lift  the  finger  and  let 
the  air  out  of  the  tube,  and  the  water  will  rush  in  at  the  lower  end  carry- 
ing the  object  with  it.  Replace  the  finger  over  the  top  of  the  tube  and 
remove  it;  the  water  will  remain  in  it  as  long  as  the  finger  is  held  firmly 
over  the  upper  end.  When  the  finger  is  removed  the  water  and  the 
object  pass  out.  The  object  may  sometimes  be  more  readily  discharged 
if  the  tube  is  rotated. 


Chapter  XIII:    Objects  of  General  Interest  95 

11.  To  Keep  Water  from  Evaporating  from  a  Cell  too  Freely  use  a  round 
cell  and  cover  it  with  a  square  cover-glass.  Apply  a  brush  wet  with 
water  to  the  slide  beneath  one  of  the  projecting  corners  of  the  cover  from 
time  to  time.  Capillary  attraction  will  draw  in  the  water  and  will  keep 
the  cell  full.  If  a  continuous  supply  of  fresh  water  is  necessary,  one 
end  of  a  loosely  twisted  cotton  thread  may  be  laid  along  one  side  of  the 
cover  and  the  other  end  of  the  thread  immersed  in  a  small  vessel  of 
water  which  stands  within  half  or  three-quarters  of  an  inch  of  the  cell. 
A  reservoir  made  from  the  bottom  of  a  shell  vial  or  homeopathic  vial 
answers  very  well;  it  may  be  cemented  to  the  slide. 

Protozoa  and  other  small  forms  may  be  kept  alive  on  a  slide  for  a 
number  of  hours  by  simply  mounting  them  in  water  under  a  cover  in  a 
cell  of  blotting  paper  which  has  been  saturated  with  water. 

12.  Deep  Cells  are  made  frequently  by  cutting  out  rings  of  paper, 
lead,  or  block-tin  with  gun  punches  and  cementing  them  to  the  slide. 
Glass  and  hard  rubber  rings  of  various  sizes  may  be  purchased  from 
dealers. 


CHAPTER  XIV 
BLOOD 

I.     EXAMINATION  OF  FRESH  BLOOD 

a)  General. — 1.  Thoroughly  clean  a  slide  and  cover,  bathe  a  finger 
in  ether-alcohol  (reagent  16,  Appendix  B),  sterilize  a  sharp  needle  by 
heating  it  in  a  flame  and  then  prick  the  finger  with  the  needle. 

2.  Place  a  small  drop  of  the  resulting  blood  on  a  slide  and  quickly 
put  on  a  cover-glass.  To  prevent  evaporation,  the  edges  of  the  cover 
may  be  surrounded  by  olive  oil  or  vaselin. 

Living  corpuscles  may  also  be  studied  in  a  drop  of  normal  saline. 

b)  Effects  of  reagents. — When  it  is  desired  to  study  the  effects  of 
reagents  on  fresh  blood  (e.  g.,  distilled  water,  1  per  cent,  tannic  acid,  etc.) 
a  drop  of  fresh  blood  is  placed  on  a  jslide,  the  cover  is  put  on  and  then 
the  blood  is  "  irrigated  "  with  the  reagent.  That  is,  a  drop  of  the  reagent 
is  placed  at  the  edge  of  the  cover  to  be  drawn  under  by  capillary  action. 
The  process  may  be  hastened  by  applying  the  edge  of  a  bit  of  blotting 
paper  to  the  opposite  edge  of  the  cover. 

c)  To  demonstrate  blood- platelets. — Place  a  small  drop  of  a  1  per 
cent,  solution  of  methyl  violet  (reagent  57,  Appendix  B)  in  normal  salt 
solution,  on  a  finger  which  has  been  cleaned  by  washing  it  in  ether- 
alcohol.  With  a  sterilized  needle  prick  the  finger  through  the  stain  and 
mount  a  drop  of  the  blood  which  exudes.  Examine  it  under  a  high 
power.     Both  platelets  and  white  corpuscles  are  stained. 

d)  Stained  preparation  of  fibrin. — Mount  a  drop  of  blood  on  a  slide 
as  in  a.  Place  it  in  a  moist  chamber  for  from  20  to  30  minutes  to 
coagulate.  Loosen  the  cover  with  a  few  drops  of  water  and  then  thor- 
oughly irrigate  the  preparation  with  water.  Drain  off  the  water,  blot 
the  preparation  with  blotting  paper  and  add  immediately  a  drop  of  a  1 
per  cent,  aqueous  solution  of  eosin  (reagent  40,  Appendix  B).  Remove 
this  after  3  minutes,  rinse  the  preparation  in  water,  then  treat  it  3  min- 
utes with  a  1  per  cent,  aqueous  solution  of  methyl  violet  (reagent  57, 
Appendix  B).  Rinse  the  preparation  in  water,  let  it  dry,  and  finally 
mount  in  balsam.* 

e)  Crystals  of  the  blood. — 

1)  Hemoglobin  Crystals. — Allow  a  drop  of  blood  to  dry  on  the  slide 
without  covering  it.  Long  rhombic  prisms  of  a  red  color  crystallize  out. 
The  blood  of  a  rat  is  best  for  demonstration.  A  more  certain  method  is 
as  follows:    To  5  c.c.  of  blood  in  a  test-tube  add  a  few  drops  of  ether 

97 


98  Animal  Micrology 

and  shake  the  mixture  vigorously  until  the  blood  becomes  laky.  Place 
a  drop  or  two  of  the  laked  blood  on  a  slide  and  allow  it  to  dry  in  the 
cold. 

2)  Hematoidin  Crystals;  reddish-yellow  crystals  (rhombic  plates). 
They  can  be  obtained  from  old  blood  extravasations  (e.  g.,  cerebral  hem- 
orrhage, corpora  lutea,  etc.)  by  teasing.    Mount  in  Canada  balsam. 

3)  Hemin  or  Teichmann's  Crystals. — To  a  small  drop  of  blood  on  a  slide 
or  a  bit  of  cloth  which  has  been  previously  saturated  with  blood,  add  a 
few  crystals  of  common  salt.  Heat  over  a  flame  until  the  mixture  has 
become  dry,  leaving  a  reddish-brown  residue.  Apply  a  cover-glass  and 
flood  the  preparation  with  as  much  acetic  acid  as  will  remain  in  place 
under  the  cover.  Heat  the  preparation  until  the  acetic  acid  boils.  After 
the  acid  has  evaporated  the  preparation  may  be  made  permanent  by 
adding  Canada  balsam.  The  crystals  are  very  small,  narrow  rhombic 
plates  of  dark  brown  color.  They  vary  in  size  and  may  lie  singly,  across 
one  another,  or  in  stellate  groups. 

The  presence  of  these  crystals  is  positive  evidence  of  the  presence  of 
blood,  hence  their  demonstration  is  of  great  importance  in  stains  or  fluid 
suspected  of  containing  blood. 

II.     COVER-GLASS  PREPARATIONS 

a)  Dry  preparations  (Ehrlich's  method). — 1.  In  this  method  the 
preparation  is  "fixed"  by  means  of  heat.  Under  one  end  of  a  copper 
bar  or  copper  triangle  (Fig.  26)  place  a  flame.  After  15  or  20  minutes  a 
given  point  on  the  bar  will  have  a  practically  constant  temperature. 
Thoroughly  clean  the  bar,  run  a  stream  of  water  along  the  top  of  it 
toward  the  flame,  and  locate  the  point  farthest  from  the  flame  at  which 
the  water  boils.  The  blood  smears  when  prepared  are  to  be  placed  film 
side  up  in  a  row  across  the  bar  about  three-fourths  of  an  inch  nearer  the 
flame  than  the  point  at  which  the  water  just  boiled.  This  will  subject 
them  to  a  temperature  of  about  120°  C. 

2.  Thoroughly  clean  and  dry  two  cover-glasses,  touch  one  to  a  drop 
of  perfectly  fresh  blood  as  it  comes  from  the  finger  or  lobe  of  the  ear  and 
instantly  drop  it  onto  the  second  cover.  The  blood  should  spread  in  a 
thin  film  between  the  covers;  if  it  does  not,  it  has  begun  to  coagulate 
and  the  preparation  will  be  inferior.  Rapidly  separate  the  covers  by 
sliding  them  apart,  wave  them  in  the  air  a  minute  to  dry  the  films,  then 
place  them  down  with  the  smear  side  uppermost.  Do  not  press  the 
covers  together  to  spread  the  blood  because  this  ruins  the  corpuscles.  If 
the  red  corpuscles  are  to  retain  their  shape  the  film  of  blood  must  be 
extremely  and  uniformly  thin.  Practice  until  you  have  prepared  such 
a  film. 


Chapter  XIV:   Blood  99 

Note. — In  the  clinical  examination  of  blood  great  care  must  be  exer- 
cised to  have  it  absolutely  fresh ;  furthermore,  the  cover-glasses  should 
be  handled  with  forceps  instead  of  by  means  of  the  fingers.  It  is  recom- 
mended that  one  pair  of  the  forceps  be  Coronet  or  spring  forceps  of  some 
kind  (Fig.  39).  The  lobe  of  the  ear  is  perhaps  the  best  region  from 
which  to  obtain  the  blood.  The  needle  with  which  the  puncture  is 
made  should  always  be  sterilized.  Wipe  away  the  first  drop  of  blood 
that  appears.  The  drop  finally  chosen  should  be  one  that  has  appeared 
immediately  after  the  spot  has  been  wiped  and  it  should  be  but  little 
larger  than  a  pin-head.  The  whole  operation  cannot  be  performed  too 
rapidly.  To  shorten  the  time  it  is  well  to  have  an  assistant  to  prick  and 
manipulate  the  ear  while  the  operator  attends  to  the  preparation  of  the 
film. 

3.  When  several  satisfactory  films  have  been  prepared,  place  them 
on  the  heated  bar  as  indicated  in  step  1.  Cover  them  to  keep  out  dust 
and  leave  them  for  from  30  to  60  minutes. 

4.  Remove  the  covers  and  stain  the  preparations  15  or  20  minutes 
with  Ehrlich's  triple  stain  (reagent  39,  Appendix  B)  by  flooding  the  film 
with  the  stain  by  means  of  a  pipette.  Rinse  off  the  surplus  stain  with 
water,  blot  the  film  with  blotting  paper  and  dry  it  by  holding  it  with 
the  edge  downward  high  above  the  flame.  When  dry,  mount  in  balsam 
on  a  slide. 

Note. — Instead  of  heating  the  preparation,  much  the  same  results 
may  be  obtained  by  subjecting  films  (prepared  as  in  step  2)  to  ether- 
alcohol  (reagent  16,  Appendix  B)  for  from  1  to  12  hours,  drying  them 
again  in  the  air  and  then  staining  as  above. 

b)  Rapid  method. — 1.  Prepare  a  film  as  above  (a  2),  only,  before  it  has 
dried  treat  it  with  concentrated  aqueous  solution  of  corrosive  sublimate 
(reagent  13,  Appendix  B). 

2.  Wash  the  preparation  thoroughly  in  water  or  in  50  per  cent,  alcohol. 

3.  Stain  for  10  minutes  in  Delafield's  or  Ehrlich's  hematoxylin  (reagent 
49  or  50,  Appendix  B),  rinse  in  70  per  cent,  alcohol  and  stain  for  20  sec- 
onds in  eosin  (0.5  per  cent,  solution  in  70  or  95  per  cent,  alcohol). 

4.  Rinse  in  95  per  cent,  and  in  absolute  alcohol  each  for  2  minutes, 
pass  through  xylol  and  mount  in  balsam. 

After  rinsing  following  staining,  some  workers  simply  blot  the  prep- 
aration with  blotting  paper,  dry  it  in  the  air  and  mount  it  in  balsam. 

III.    ENUMERATION  OF  BLOOD  CORPUSCLES 

"  The  instrument  used  is  the  hemocytometer  (Fig.  37).  Obtain  a  drop 
of  blood  from  the  lobe  of  the  ear  or  from  the  finger.  Fill  the  clean 
pipette  of  the  hemocytometer  to  the  mark  1  by  careful  suction.  (If  the 
blood  is  drawn  beyond  the  1  mark,  blow  it  out  immediately,  clean  the 


100 


Animal  Micrology 


tube  and  repeat  the  operation.)  Wipe  the  blood  from  the  outside  of  the 
pipette  and  draw  in  sufficient  Toisson's  solution  to  make  the  level  of  the 
combined  liquids  stand  precisely  at  the  mark  101. 

Toisson's  solution: 

Sodium  sulphate 8.0     grams 

Sodium  chloride 1.0      gram 

Neutral  glycerin 30.0     c.c. 

Methyl  violet,  5b 0.025  gram 

Distilled  water 160.0      c.c. 

"  Mix  the  blood  thoroughly  with  the  solution  by  shaking  the  tube  for 
a  few  minutes.     The  blood  is  thus  diluted  100  times. 

"  Blow  out  a  drop  of  the  liquid  to  remove  the  unmixed  solution  remain- 
ing in  the  capillary  tube.    Have  the  counting  disk  and  cover-glass  per- 


0.100mm. 

0 

C.  Zeiss 
Jena. 

Fig.  37.— Hemocytometer. 

a,  view  of  slide  from  above;  ft,  view  of  slide  from  one  side;  c,  counting-disk  which  lies 
at  the  center  of  B ;  E,  bead  for  mixing ;  M,  mouthpiece. 

fectly  clean.  Allow  a  drop  of  the  diluted  blood  to  flow  onto  the  disk  and 
place  the  cover-glass  over  the  drop.  The  cell  of  the  disk  must  be  entirely 
filled  by  the  drop  of  blood. 

"  Examine  the  preparation  under  a  high  power  of  the  microscope,  and 
count  the  number  of  red  corpuscles  in  20  to  40  small  squares;  of  those 
corpuscles  which  happen  to  lie  on  the  boundary  line,  count  the  ones  that 
lie  only  in  the  upper  and  on  the  left  sides  of  each  square.  Take  the 
average  number  in  a  square  and  calculate  the  number  of  corpuscles  in  a 
cubic  millimeter  of  blood. 

"  The  depth  of  the  entire  cell  is  0.1  mm.,  the  area  of  each  small  square 
is  too  sq.  mm.,  consequently  the  volume  of  blood  in  each  square 
column  is  tuW  c.  mm.,  or  1  cubic  millimeter  of  diluted  blood  would 
contain  4,000  times  the  average  number  in  a  square.     One  cubic  milli- 


Chapter  XIV:   Blood  101 

meter  of  undiluted  blood  contains  100  times  as  many,  or  400,000  times 
the  number  in  one  square.     What  result  do  you  obtain? 

"After  finishing  the  count,  clean  the  pipette  by  successively  drawing 
into  and  expelling  from  it  water,  alcohol,  and  finally  ether.  Do  not  blow 
through  it,  but  cause  the  ether  to  evaporate  by  sucking  air  through  the 
tube.  For  counting  the  white  corpuscles  use  the  large  pipette  and  dilute 
the  blood  10  times  with  one-third  of  1  per  cent,  glacial  acetic  acid.  The 
acid  destroys  the  red  corpuscles  and  thus  the  white  corpuscles  are  more 
readily  seen.  Proceed  in  the  same  manner  as  for  red  corpuscles."  (From 
Laboratory  Outlines  for  Physiology,  by  Guyer  and  Pauli.) 

IV.   OBSERVATION  OF  THE  BLOOD  CURRENT 

a)  Circulation  in  the  web  of  a  frog's  foot. — Wind  a  long  strip  of 
cheese  cloth  around  a  frog  stretched  out  upon  a  narrow  piece  of  thin  board, 
leaving  one  hind  foot  exposed.  Soak  the  cloth  in  water  in  order  to  keep 
the  animal's  skin  moist.  Pin  the  extended  foot  onto  a  ring  of  cork  in 
such  a  way  that  the  web  between  the  toes  is  stretched  over  the  opening 
in  the  cork.  Examine  under  the  microscope.  If  the  preparation  is  favor- 
able leucocytes  may  perhaps  be  seen  penetrating  the  walls  of  the  vessel 
(diapedesis)  and  passing  into  the  surrounding  tissues. 

b)  Circulation  in  the  mesentery.  Inflammation. — Immobilize  a  frog 
(the  male  is  better)  by  injecting  a  few  drops  of  a  1  per  cent,  solution  of  cur- 
are into  one  of  the  dorsal  lymph  sacs.  Curare  paralyses  the  nerve  endings. 
After  waiting  20  minutes  for  the  curare  to  be  absorbed  into  the  circula- 
tion, cut  open  the  abdominal  wall  for  a  short  distance  along  the  left  side 
and  draw  out  several  loops  of  the  intestine.  Pin  out  a  favorable  area  of 
mesentery  over  a  cork  ring,  and,  after  covering  it  with  a  cover-glass, 
examine  under  the  microscope.  Keep  the  parts  moistened  with  normal 
salt  solution.  Such  a  preparation  is  especially  favorable  for  studying 
the  migrations  of  leucocytes  through  the  walls  of  the  vessels.  Do  not 
have  the  mesentery  stretched  too  tightly  or  the  circulation  will  cease. 
After  a  time  the  phenomena  of  inflammation  may  readily  be  observed. 
It  is  hastened  if  some  irritant  (e.  g.,  a  drop  of  creosote)  is  applied  to  the 
mesentery. 

MEMORANDA 

1 ,  For  Demonstration  of  the  Different  Granules  of  Leucocytes,  etc.,  see 
Appendix  C,  I,  under  the  general  topic  of  blood. 

2.  To  Study  Blood  in  Sections,  ligate  a  small  vessel  in  two  places  to 
keep  in  the  corpuscles,  then  remove  the  piece  so  prepared  and  fix  it  in 
Gilson's  fluid  (reagent  15,  Appendix  B)  or  Hermann's  fluid  (reagent  26, 
Appendix  B).  Imbed  in  paraffin  and  cut  thin  sections.  Stain  material 
fixed  in  Gilson  or  other  corrosive  sublimate  reagents  by  the  hematoxylin 


102  Animal  Micrology 

eosin  method  (reagents  49,  40,  Appendix  B)  or  with  the  Ehrlich-Biondi 
stain  (38,  Appendix  B).  The  blood  fixed  in  Hermann's  fluid  may  be 
stained  by  the  saffranin-gentian  violet  method  (66,  Appendix  B). 

3.  Amoeboid  Movements  in  Leucocytes  may  readily  be  observed  in  blood 
(preferably  amphibian)  which  has  been  mounted  on  a  slide  in  very 
slightly  warmed  normal  saline.  Place  a  hair  under  the  cover-glass  and 
seal  the  edges  of  the  latter  with  vaselin  or  melted  paraffin.  For  continu- 
ous study  of  the  white  corpuscles  of  warm-blooded  animals  a  warm  stage 
of  some  kind  is  necessary,  to  keep  the  temperature  of  the  blood  near  the 
temperature  of  the  body. 

4.  Feeding  Leucocytes. — Rub  up  sufficient  India  ink  in  a  few  drops  of 
normal  saline  to  make  a  grayish  fluid.  With  fine  scissors  make  an  inci- 
sion into  one  of  the  dorsal  lymph  sacs  of  a  frog  (parallel  to  and  close 
beside  the  urostyle).  Introduce  a  capillary  pipette  into  the  wound  and 
obtain  a  small  drop  of  lymph.  Mix  it  on  a  slide  with  a  drop  or  two  of 
the  prepared  ink.  After  placing  a  hair  across  the  field  put  on  a  cover- 
glass  and  seal  the  edges  with  vaselin  or  melted  paraffin.  Under  a  high 
power  of  the  microscope  the  cells  may  be  seen  engulfing  the  colored 
particles. 

5.  Wright's  Stain  for  Blood. — To  prepare  the  stain  make  a  0.5  per  cent, 
solution  of  sodium  bicarbonate  in  distilled  water  and  add  to  it  1  per  cent, 
of  methylen  blue  (Grubler).  Subject  the  mixture  to  steam  in  an  ordi- 
nary steam  sterilizer  (e.  g.,  Arnold;  not  a  pressure  sterilizer  or  a  water- 
bath)  for  one  hour.  When  the  mixture  is  cool,  without  filtering,  pour  it 
into  a  large  dish.  Prepare  a  1  per  cent,  aqueous  solution  of  Grtibler's 
yellowish  eosin  (soluble  in  water)  and,  with  constant  stirring,  add  it  to 
the  methylen-blue  solution  until  the  blue  color  is  replaced  by  purple,  and 
a  yellowish  scum  with  metallic  luster  forms  on  the  surface  of  the  mixture, 
while  a  finely  granular  black  precipitate  appears  in  suspension.  The 
proportions  required  will  be  about  500  parts  of  the  eosin  to  100  parts  of 
the  methylen  blue  solution.  Collect  the  precipitate  on  a  filter,  dry  it 
thoroughly,  make  a  5  per  cent,  solution  in  pure  methylic  alcohol.  To 
prevent  the  alcohol  from  evaporating  keep  the  bottle  containing  the  solu- 
tion tightly  stoppered.  Should  precipitation  occur,  filter  the  stain  and 
add  a  small  quantity  of  methyl  alcohol. 

Mallory  and  Wright  in  their  Pathological  Technique,  p.  374,  give 
the  following  summary  of  the  method  for  staining  blood  films: 

"1.  Make  films  of  the  blood,  spread  thinly,  and  allow  them  to  dry  in 
the  air. 

"2.  Cover  the  preparation  with  the  staining  fluid  for  one  minute. 

"  3.  Add  to  the  staining  fluid  on  the  preparation  sufficient  water,  drop 
by  drop,  until  a  delicate,  iridescent,  metallic  scum  forms  on  the  surface. 
Allow  this  mixture  to  remain  on  the  preparation  for  two  or  three  minutes. 


Chapter  XIV:   Blood  103 

"4.  Wash  in  water,  preferably  in  distilled  water,  until  the  film  has  a 
pinkish  tint  in  its  thinner  or  better-spread  portions  and  the  red  cor- 
puscles acquire  a  yellow  or  pink  color. 

"  5.  Dry  between  filter-paper  and  mount  in  balsam.  The  preparations 
retain  their  colors  as  long  as  any  preparations  stained  with  anilin  dyes. 

u  Unstained  blood  films  may  be  kept  for  some  weeks  without  impair- 
ment of  their  staining  properties.  Films  months  old  will  probably  not 
give  good  results." 

6.  For  Malarial  Parasites  Wright's  stain  (memorandum  5)  is  excellent. 
It  yields  the  so-called  Romanowsky  stain;  the  color  of  the  chromatin 
varies  from  lilac  to  very  dark  red,  while  the  body  of  the  parasite  stains 
blue.  A  full  account  of  the  method  will  be  found  in  Mallory  and 
Wright's  Pathological  Technique,  p.  421. 


CHAPTER  XV 

BACTERIA 

No  attempt  is  made  here  to  give  even  an  elementary  account  of 
bacteriological  technique.  Only  such  phases  of  the  work  as  are  concerned 
with  the  immediate  microscopical  examination  of  bacteria  are  touched 
upon,  and  these  chiefly  to  afford  some  practice  in  this  kind  of  manipula- 
tion. For  special  technique,  identification,  or  descriptions  of  apparatus 
and  accessories,  the  student  is  referred  to  standard  textbooks. 

BACTERIAL   EXAMINATION 

Bacteria  when  prepared  for  microscopical  examination  are  in  the 
form  of 

A.  Cover-glass  preparations, 

B.  Bacteria  in  tissues  (section  method),  or 

C.  Hanging-drop  preparations. 

A.  Cover-Glass  Preparations 
I.  Killing  and  fixing. 

1.  From  Fluid  Media  (e.  g.,  bouillon,  milk,  water,  saliva,  blood,  pus 
etc.). — Sterilize  a  platinum  wire  loop  by  heating  it  red  hot  in  a  flame. 
When  cool,  touch  the  loop  to  the  culture  and  spread  the  adherent 
bacteria  in  a  thin  film  over  the  surface  of  a  cover-glass  which  has  been 
sterilized  in  a  flame.  After  the  film  has  dried  in  the  air,  kill  and  fix  the 
bacteria  to  the  cover  by  passing  it  three  times,  film  side  uppermost, 
through  the  apex  of  a  flame.  Each  time  should  not  exceed  half  a  second. 
Prepare  several  films  from  a  given  material.  Coronet  or  similar  forceps 
(Figs.  38,  39)  should  be  used  for  handling  such  films,  because  the  cover- 
glass  can  be  left  in  them  through  the  entire  operation  of  fixing  and 
staining. 

If  a  platinum  loop  is  not  at  hand  a  second  cover-glass  may  be  used 
to  spread  the  smear.  The  first  cover-glass  is  held  in  a  pair  of  cover-glass 
forceps  and  the  second  cover-glass  is  dropped  on  to  it.  The  glasses  are 
then  rapidly  drawn  apart  with  a  sliding  motion  by  means  of  forceps. 
The  glasses  should  not  be  pressed  tightly  together.  Proficiency  in 
making  such  preparations  is  gained  only  after  considerable  practice. 
The  chief  secret  in  making  a  good  preparation  is  to  get  the  films  extremely 
thin  and  evenly  distributed. 

2.  From  Solid  Media  (gelatin,  agar,  meat,  potato,  animal  tissues  and 
organs,  etc.). — The  procedure  is  the  same  as  for  1,  except  that  a  drop  of 

105 


106  Animal  Micrology 

sterilized  water  or  bouillon  is  put  on  the  cover-glass  to  facilitate  the 
spreading  of  the  bacteria  in  a  film  over  the  cover. 

II.  Staining  and  Mounting. 

1.  Gentian  violet  (memorandum  3  a)  5  minutes.  The  cover-glass  is 
left  in  the  forceps,  film  side  up,  and  the  film  flooded  with  the  staining 
fluid. 


Fig.  38.— Cornet's  Cover-Class  Forceps. 

2.  Rinse  in  water. 

3.  Gram's  solution  (memorandum  3/)  until  the  color  becomes  black 
(2  to  3  minutes). 

4.  Ninety-five  per  cent,  alcohol  until  the  violet  color  has  almost  com- 
pletely disappeared. 

5.  Rinse  in  water  and  examine  by  placing  the  cover-glass  film  side 
downward  on  a  slide.  Only  a  thin  film  of  water  should  remain  between 
the  slide  and  the  cover.  Remove  surplus  water  by  means  of  blotting 
paper.  If  a  prolonged  examination  is  to  be  made,  water  lost  by  evapora- 
tion must  be  replaced  by  occasionally  placing  a  small  drop  of  water  at 


Fig.  39.— Stewart's  Steel  Wire  Cover-Glass  Forceps. 

the  edge  of  the  cover.  In  ordinary  work  the  final  inspection  is  frequently 
made  at  this  stage.  If  a  permanent  preparation  is  desired,  however, 
proceed  with  the  following  steps : 

6.  If  the  bacteria  are  well  stained,  a  counterstain  of  Bismarck  brown 
(memorandum  3d,  sol.  2)  may  be  added  (5  to  10  seconds).  This  step  may 
be  omitted. 

7.  Absolute  alcohol,  10  to  15  seconds. 

8.  Xylol. 

9.  Xylol-balsam. 

Note. — In  staining,  if  the  cover-glass  is  warmed  over  a  flame  some 
15  or  20  seconds  until  the  stain  steams,  the  action  of  the  stain  is  usually 
more  intense  and  more  rapid.    Boiling,  however,  must  be  avoided. 


Chapter  XV:   Bacteria  107 

B.  Bacteria  in  Tissues 

Tissues  may  be  fixed  and  hardened  (e.  g.,  Gilson's  fluid,  Appendix  B, 
reagent  15;  or  Zenker's,  reagent  6;  or  formalin,  reagent  17)  in  the 
ordinary  way,  and  sections  made  by  the  usual  methods.  Where  practi- 
cable paraffin  sections  are  preferable  to  celloidin  sections,  because  the 
celloidin  tends  to  hold  the  stain  and  thus  obscure  the  bacteria.  Sections 
should  be  fixed  to  the  slide  (paraffin  by  albumen  fixative,  celloidin  by 
ether  vapor). 

Bacteria  which  do  not  stain  by  the  Gram  method  (memorandum  3/) 
or  the  tubercle  bacillus  method  (memorandum  Se)  are  difficult  to  demon- 
strate, because  it  is  hard  to  stain  them  so  as  to  differentiate  them  from 
the  tissues  in  which  they  lie;  furthermore,  most  of  them  easily  lose  what- 
ever stain  they  may  have  taken  up.  Ldffler's  alkaline  methylen  blue 
(memorandum  Sb)  is,  perhaps,  the  most  useful  stain  for  these  organisms. 

Methylen  Blue  Stain  for  Bacteria  in  Tissues. — 1.  Stain  sections  (paraffin) 
30  minutes  to  24  hours. 

2.  Acetic  acid  (1  to  1,000  of  water)  10  to  20  seconds. 

3.  Rinse  in  absolute  alcohol  20  to  30  seconds. 

4.  Xylol. 

5.  Xylol-balsam. 

With  celloidin  sections  substitute  95  per  cent,  alcohol  for  absolute 
(step  3),  then  treat  with  xylol  or,  better,  carbol-xylol  until  sections  are 
clear.    Mount  in  xylol-balsam. 

Anilin  gentian  violet,  methyl  blue,  methyl  violet,  or  fuchsin  (memo- 
randum 3  a),  also  carbol -fuchsin  (memorandum  3  c)  may  be  used  in  the 
same  way. 

Gram's    Method    for    Bacteria    in    Tissues    (Weigert's   modification). — 
1.  Stain  sections  (any  kind)  in  lithium  carmine  2  to  5  minutes. 
Lithium  Carmine  (Orth's)  : 

Carmine 2.5  to  5  grams. 

Carbonate  of  lithium,  saturated  aqueous  solution    100  c.c. 

Thymol       , a  crystal  or  two. 

Filter. 

2.  Anilin  gentian  violet  5  to  20  minutes  (celloidin  sections  should  first 
be  dehydrated  in  95  per  cent,  alcohol  and  affixed  to  the  slide  with  ether 
vapor). 

3.  Rinse  in  normal  saline. 

4.  Gram's  solution  (memorandum  3/)  1  to  2  minutes. 

5.  Rinse  in  water. 

6.  Blot  sections  with  filter  paper  to  remove  as  much  water  as  possible. 

7.  Anilin  oil,  several  changes.  The  oil  dehydrates,  and  at  the  same 
time  decolorizes  the  celloidin. 

8.  Xylol,  several  changes. 

9.  Xylol-balsam. 


Fig.  40.— Culture  Slide. 


108  Animal  Micrology 

C.  Hanging-Drop  Preparations 

1.  A  slide  with  a  concave  center  is  used  (Fig.  40).  With  a  fine-pointed 
brush  paint  a  narrow  strip  of  vaselin  around  the  margin  of  the  concavity. 
The  vaselin  makes  the  cover-glass  stick  to  the  slide  and  also  prevents 
evaporation. 

2.  Place  a  small  drop  of  the  fluid  containing  bacteria  in  the  center  of 
the  cover-glass.     If  the  bacteria  to  be  examined  are  on  a  solid  medium 

the  "drop"  should  be  made  by 
Ml  llMBleiiai  mixing  a  small  portion  of  the 
growth  with  a  drop  of  bouillon, 
normal  saline,  or  serum.     Place 

the  cover-glass,  drop  downward,  over  the  depression  in  the  slide  and 

press  it  down  well  into  the  vaselin. 

3.  Use  only  a  small  opening  in  the  diaphragm  when  examining  the 
bacteria,  in  order  to  get  as  much  contrast  by  refraction  as  possible. 
Focus  first  with  a  medium-power  dry  objective  on  the  edge  of  the  drop, 
then  employ  the  oil  immersion.  Such  unstained  organisms  are  fre- 
quently difficult  to  find  and  there  is  great  danger  of  breaking  the  cover- 
glass  with  the  objective. 

Hanging-drop  preparations  are  used  mainly  in  determining  the 
motility  of  bacteria,  or  in  the  study  of  spore  formation.  For  the  latter 
purpose,  the  slide  and  cover-glass  must  be  carefully  sterilized  and  the 
sealing  with  vaselin  complete.  The  preparation  may  then  be  placed  on 
a  warm  stage  or  in  an  incubator  and  examined  from  time  to  time. 

MEMORANDA 

1.  The  Main  Points  to  Be  Observed  in  the  Microscopical  Examination  of 
Bacteria  are  as  follows:  (1)  form  of  the  individual,  whether  spherical 
(coccus),  spiral  (spirillum),  or  rodlike  (bacillus)  with  end  square,  pointed, 
or  rounded;  (2)  uniformity  in  size;  (3)  the  arrangements  of  individuals 
whether  single  (micrococci,  etc.),  in  pairs  (e.  g.,  diplococci),  in  chains 
(e.  g.,  streptococci),  groups  of  four  (e.  g.,  tetracocci),  cubical  groups  of 
eight  or  more  (sarcinae),  or  small  grape-like  bunches  of  various-sized 
cocci  (staphylococci);  (4)  presence  or  absence  of  cell-wall,  gelatinous 
capsule,  etc.;  (5)  motility  in  living  forms  (do  not  confuse  with  Brownian 
movement);  (6)  reaction  to  stains;  (7)  presence  of  spores  which  are  rec- 
ognizable as  bright,  highly  refractive  rounded  bodies. 

2.  Material  for  the  Demonstration  of  Bacteria  (coccus,  bacillus,  spirillum, 
and  beggiatoa  forms)  will  be  found  in  abundance  in  foul  water,  espe- 
cially when  contaminated  with  sewage.  By  scraping  the  inside  of  the 
cheek  such  forms  as  Leptothrix  may  often  be  found.  Make  a  cover- 
glass  preparation;  kill  and  fix  in  the  flame  in  the  ordinary  way;  stain  in 


Chapter  XV:   Bacteria  109 

methyl  violet,  gentian  violet,  or  fuchsin  (basic)  and,  if  desired,  counter- 
stain  lightly  with  Bismarck  brown;  examine  in  water  or  dehydrate  in 
absolute  alcohol,  clear  in  xylol  and  mount  in  balsam. 

To  demonstrate  bacteria  in  tissues,  a  mouse  may  be  inoculated  with 
anthrax,  and  paraffin  sections  of  the  spleen  prepared.  Stain  by  the 
gentian  violet  method. 

3.  Some  of  the  Most  Important  Stains  for  Bacteria  are  as  follows: 

a)  Anilin  water  solution  of  gentian  violet  (Koch-Ehrlich's). — 

Gentian  violet,  saturated  alcoholic  solution  ...      10  c.c. 
Anilin  water  (see  Appendix  B,  reagent  29)    .     .     .    100  c.c. 

After  shaking  it,  the  mixture  should  be  set  aside  for  24  hours  because  of 
the  precipitation  which  takes  place  soon  after  making.  Solutions  of 
fuchsin  (basic)  and  methyl  blue  are  made  in  the  same  way.  These  solu- 
tions begin  to  decompose  after  about  10  days  and  must  then  be  freshly 
prepared.  They  yield  good  results  with  many  species  of  bacteria.  The 
gentian  violet,  particularly,  is  widely  used  in  connection  with  Gram's 
method  (see  /). 

b)  Alkaline  methdlen  blue  (Loeffler's). — 

Methylen  blue,  saturated  alcoholic  solution      .     .      30  c.c. 
Caustic  potash,  aqueous  solution  (1:  10,000).     .     .    100  c.c. 

This  stain  keeps  well  and  is  one  of  the  most  widely  used  of  the  general 
stains.  It  is  especially  serviceable  in  staining  the  bacillus  of  diphtheria 
or  of  glanders. 

c)  Carbol- fuchsin  (Ziehl-Neelson's). — 

Fuchsin,  saturated  alcoholic  solution 10  c.c. 

Carbolic  acid,  5  per  cent,  aqueous  solution    ...      90  c.c. 
This  stain  keeps  well,  stains  powerfully,  and  can  be  used  on  many  forms 
of  bacteria. 

d)  Neisser's  method  for  the  diagnosis  of  Diphtheria. — 
Solution  I. 

Methylen  blue  (Grtlbler's) 1  gram 

Alcohol,  96  per  cent 20  c.c. 

Distilled  water  (add  after  the  methylen  blue  has 

dissolved  in  the  alcohol) 950  c.c. 

Glacial  acetic  acid 50  c.c. 

Solution  II. 

Bismarck  brown 1  gram 

Distilled  water  (should  be  boiling  when  the  Bis- 
marck brown  is  added) 500  c.c. 

Cover-glass  preparations  are  stained  for  from  2  to  3  seconds  in  Solution 
I,  rinsed  in  distilled  water,  placed  in  Solution  II  for  from  3  to  5  seconds, 


110  Animal  Micrology 

rinsed  again  in  water,  and  examined  in  the  ordinary  way.  The  bacteria 
of  virulent  diphtheria  should  appear  as  pale-brown  rods,  some  of  which 
show  at  one  or  both  ends  bluish-black  oval  bodies  of  greater  diameter 
than  the  rod.  Such  dark  bodies  will  not  be  seen  in  the  pseudo-diph- 
theria bacilli. 

The  bacilli  must  have  been  grown  for  from  12  to  18  hours  on  Loff- 
ler's  blood-serum  which  is  a  mixture  of  glucose  bouillon  1  part  and  beef- 
blood  serum  3  parts.  The  mixture  is  run  into  test-tubes  and  coagulated 
at  100°  C.j  the  tube  should  be  tilted  to  one  side  to  give  a  slanting  surface 
for  culture  purposes.  The  formula  for  glucose  bouillon  is  as  follows: 
dry  glucose,  10  grams;  Liebig's  extract  of  beef,  3  grams;  peptone,  10 
grams;  sodium  chloride,  5  grams;  water  1,000  c.c. 

e)  Gabbefs  solution  for  demonstrating  tubercle  bacilli. — 

Methylen  blue 1  to  2  grams 

Distilled  water 75  c.c. 

Concentrated  sulphuric  acid 25  c.c. 

The  acid  decolorizes,  while  the  methylen  blue  serves  as  a  contrast  stain. 
The  solution  acts  rapidly.  A  modification  of  the  method  to  be  com- 
mended is  first  to  stain  the  preparation  with  carbol-fuchsin  (see  c)  by 
warming  the  stain  on  the  slide  until  it  steams,  rinsing  in  water  and 
then  proceeding  with  the  methylen-blue  solution.  Smegma,  leprosy, 
and  syphilis  bacilli  are  also  stained  by  this  method.  Tubercle  bacilli 
are  also  stained  by  Gram's  method  (see  /).  To  examine  sputum  for 
tubercle  bacilli,  the  sputum  is  carefully  inspected  for  small  yellowish- 
white  cheesy  masses  varying  in  size  from  the  diameter  of  a  pin-head  to 
that  of  a  small  pea.  Very  thin  smear  preparations  (see  A)  are  made 
from  such  masses. 

/)  Gram's  method. — 
Gram's  solution. 

Iodine  crystals 1  gram 

Iodide  of  potassium 2  grams 

Distilled  water 300  c.c. 

The  preparations  are  first  stained  in  anilin  gentian  violet  (memorandum 
3a),  and  then  immersed  in  Gram's  solution  for  from  1  to  2  minutes. 
They  are  then  rinsed  in  alcohol  until  the  violet  color  is  no  longer  visible 
to  the  naked  eye.  To  decolorize  them  sufficiently,  it  may  be  necessary 
to  treat  them  again  with  the  iodine  solution.  Finally  rinse  in  water  and 
•examine,  or  if  a  permanent  preparation  is  desired,  rinse  in  absolute  alco- 
hol, transfer  to  xylol,  and  mount  in  balsam.  If  the  preparations  are 
from  cultures,  it  should  be  borne  in  mind  that  the  method  works  well 
only  when  applied  to  bacteria  from  actively  growing  cultures;  old  cul- 
tures seldom  yield  satisfactory  results. 


Chapter  XV:   Bacteria 


111 


Pathogenic  Bacteria  Stained 
by  Gram's  Method 
Bacillus  aerogenes  capsulatus 
Bacillus  of  anthrax 
Bacillus  diphtheriae 
Bacillus  of  malignant  edema 
Bacillus  of  tetanus 
Bacillus  of  tuberculosis 
Micrococcus  tetragenus 
Pneumococcus 

Staphylococcus  pyogenes  aureus 
Staphylococcus  pyogenes  albus 
Streptococcus  pyogenes 

Streptococcus  capsulatus 


Pathogenic  Bacteria  Decolorized 
by  Gram's  Method 
Bacillus  of  bubonic  plague 
Bacillus  of  chancroid 
Bacillus  coli  communis 
Bacillus  of  dysentery 
Bacillus  of  glanders 
Bacillus  of  influenza 
Bacillus  mucosus  capsulatus 
Bacillus  proteus 
Bacillus  pyocyaneus 
Bacillus  of  typhoid 
Diplococcus  intra  cellularis 

meningitidis 
Gonococcus 
Spirillum  of  Asiatic  cholera 


4.  Staining  Spores  (Abbott's  method). — Prepare  a  cover-glass  smear  in 
the  usual  way.  Apply  the  stain  (e.  g.,  methylen  blue)  and  hold  the 
cover-glass  over  a  flame  until  the  liquid  steams.  Repeat  the  heating 
several  times,  but  do  not  boil  continuously.  Rinse  the  cover-glass  in 
water  and  then  decolorize  the  preparation  in  a  0.3  per  cent,  solution  of 
hydrochloric  acid  in  95  per  cent,  alcohol,  until  all  color  visible  to  the 
naked  eye  has  disappeared.  Wash  in  water.  If  a  counterstain  is  de- 
sired, stain  for  from  8  to  10  seconds  in  anilin-fuchsin  solution.  Rinse 
in  water  and  mount  in  the  usual  way.    The  spores  are  stained  blue. 

5.  Staining  Flagella  (Bunge's  modification  of  Loeffler's  method). — The 
locomotor  organs  of  motile  bacteria  are  long,  hairlike  prolongations  (1  to 
many)  termed  flagella.  Special  methods  of  staining  are  necessary  for 
their  demonstration. 

Make  thin  cover-glass  smears  of  an  18-hour  culture  which  contains 
motile  forms.     Dry  and  fix  in  the  ordinary  way. 

The  mordant. — 

Ferric  chloride,  aqueous  solution  (1:20)     ....    25  c.c. 

Alum,  saturated  aqueous  solution      ......    75  c.c. 

Shake  well  and  add, 

Fuchsin  (basic),  saturated  aqueous  solution  ...    10  c.c. 


Filter  and  allow  to  stand  for  some  time  before  using.  Treat  the 
smear  for  5  minutes  with  this  preparation,  gently  warming  by  holding 
it  high  above  a  flame.  The  fluid  must  not  boil.  Rinse  in  water,  then 
stain  faintly  with  carbol-fuchsin.  Repeat  the  process  until  a  successful 
result  is  obtained.    Mount  in  the  usual  wav. 


\ 


CHAPTER  XVI 

SOME  EMBRYOLOGICAL  METHODS:    THE  CHICK,  SECTIONS 

AND  "IN  TOTO"  MOUNTS;  AMPHIBIA;  FISH; 

MAMMALS;  OTHER  FORMS 

THE  CHICK 

The  hen  or  an  artificial  incubator  is  necessary.  In  many  ways 
the  latter  is  more  convenient  as  it  may  be  kept  in  the  laboratory 
and  is  ready  at  all  seasons  of  the  year.  There  are  many 
kinds  of  good  incubators  on  the  market  at  present  which  may  be 
had  for  a  small  sum. 

Whatever  method  of  incubation  is  employed  the  eggs  must  be 
fresh  and  must  not  have  been  subjected  to  rough  handling.  The 
date  and  hour  at  which  incubation  is  to  begin  should  be  written 
on  the  shell  of  each  egg  in  ink.  If  late  stages  of  development 
are  desired  the  egg  must  be  turned  every  few  days.  All  products 
of  combustion  from  the  lamp  or  burner  should  be  kept  from  the 
eggs  and  the  supply  of  fresh  air  and  moisture  carefully  main- 
tained. The  temperature  should  be  maintained  at  38°  C. 
(100.4°  F.).  Should  it  rise  above  40°  C.  embryos  will  be 
destroyed. 

Prepare  at  least  5  embryos  as  directed  in  the  practical  exer- 
cise ;  2  for  in  toto  preparations  and  3  for  sections. 

1.  Place  an  egg  which  has  been  incubated  for  between  46 
and  54  hours,  while  it  is  yet  warm,  in  a  vessel  which  contains 
sufficient  normal  saline  warmed  to  38°  C.  to  cover  the  egg.  In 
the  chick  the  embryo  always  makes  its  appearance  as  a  germinal 
disc  or  cicatricula,  as  it  is  termed,  situated  on  one  side  of  the 
yolk,  which  is  the  real  egg  of  the  hen,  the  white  being  simply  a 
nutritive  mass  added  in  the  oviduct.  This  disc  or  blastoderm  in 
the  early  stages  of  incubation  always  turns  uppermost  no  matter 
in  what  position  the  egg  may  be  placed.  Moreover,  it  has  been 
found  that  the  embryo  in  nearly  every  instance  lies  in  such  a 
position  that  when  the  blunt  end  of  the  egg  is  toward  the  left, 

113 


114  Animal  Micrology 

the  head  of  the  chick  is  directed  away  from  the  operator.  This 
fact  affords  a  very  reliable  means  of  orienting  the  embryo, 
especially  in  the  very  young  stages  when  the  anterior  and  pos- 
terior ends  are  not  easily  recognized  by  the  observer. 

2.  Break  through  the  shell  at  the  broad  end  over  the  air 
chamber  by  tapping  it  sharply  and  let  out  the  air,  or  the  broad 
end  will  tilt  up. 

3.  Begin  at  the  hole  made  in  the  end  and  with  blunt  forceps 
remove  the  shell  and  shell  membrane  bit  by  bit  from  the  upper 
surface  of  the  egg  until  the  embryo  comes  plainly  into  view. 
Remove  with  a  pipette  the  thin  layer  of  albumen  which  lies  above 
the  blastoderm. 

4.  With  as  little  agitation  of  the  liquid  in  the  vessel  as  possi- 
ble, by  means  of  fine  scissors  cut  rapidly  around  the  blastoderm 
just  outside  the  vascular  area. 

5.  Carefully  float  the  blastoderm  into  a  thin  watch-glass,  keep- 
ing it  as  flat  as  possible.  Shake  it  gently  to  remove  the  piece  of 
vitelline  membrane  covering  it,  or  any  yolk  which  may  adhere. 
The  aid  of  a  needle  may  be  necessary  to  remove  the  vitelline 
covering. 

6.  With  a  pipette  remove  all  fluid  from  the  watch-glass,  leav- 
ing the  blastoderm  to  become  dry  enough  to  adhere  to  the  glass, 
but  take  great  pains  that  the  embryo  itself  does  not  become  dry. 
If  the  edges  are  not  thus  kept  down  they  curl  up  and  obscure 
the  embryo  when  the  iixing  fluid  is  added.  (Some  workers 
employ  a  ring  of  paper  to  hold  down  the  edges  of  the  blastoderm.) 

7.  Carefully  add  Gilson's  fluid  (reagent  15,  Appendix  B)  until 
the  embryo  is  completely  immersed.  The  fluid  should  be  allowed 
to  act  for  from  2  to  3  hours. 

8.  Wash  in  repeated  changes  of  50  per  cent,  alcohol  to  which 
tincture  of  iodine  has  been  added  (see  caution  1  under  reagent 
13,  Appendix  B),  and  stain  in  Borax-carmine  (reagent  32, 
chap,  i)  for  24  hours  (Conklin's  hematoxylin  may  be  used  if  pre- 
ferred; see  reagent  48,  Appendix  B). 

9.  Wash  the  object  in  water  and  transfer  it  through  35  and 
50  per  cent,  alcohol  to  acid  alcohol,  leaving  it  30  minutes  in  each. 


Chapter  XVI:   Some  Embryological  Methods         115 

Decolorize  until  the  embryo  becomes  bright  scarlet  in  color,  then 
wash  in  70  per  cent,  alcohol  and  leave  it  there  until  ready  to 
proceed. 

10.  Transfer  the  object  through  95  per  cent.  (1  hour)  abso- 
lute alcohol  (2  hours)  to  xylol,  where  it  should  remain  about  2 
hours  or  until  it  ceases  to  appear  opaque. 

Mo  ant  two  embryos  entire,  one  with  the  ventral,  the  other  with 
the  dorsal  side  uppermost.  Put  bits  of  broken  cover-glass  under 
the  edges  of  the  cover  to  avoid  crushing  them. 

The  three  remaining  embryos  are  to  be  so  sectioned  (step  llff.), 
that  the  student  will  have  a  complete  series  of  sections  in 
each  of  the  three  different  planes  of  the  body  with  reference  to 
the  axis  of  the  spinal  cord:  viz.,  transverse,  sagittal,  and  frontal. 
Read  carefully  memorandum  12  on  orientating  serial  sections. 

Caution. — Before  sectioning  any  embryo  always  make  an 
outline  drawing  of  the  entire  embryo,  then  rule  lines  across  the 
drawing  parallel  to  the  plane  of  section.  Unless  this  is  done 
great  difficulty  will  be  experienced  frequently  in  understanding 
the  sections. 

11.  Infiltrate  the  embryo  with  paraffin  in  the  usual  manner  by 
leaving  it  in  melted  paraffin  for  2  or  3  hours.  A  softer  paraffin 
(melting  at  43°  C.)  than  has  been  used  heretofore  may  be 
employed  and  the  sections  cut  thicker  (15  to  20  microns). 

12.  Imbed  and  cut  in  the  usual  way  (chap.  v).  Mount  the 
entire  series. 

MEMORANDA 

1.    For  the  Average  Course  in   Embryology  of  the   Chick   the   following 
mounted  stages  are  the  most  useful : 
I.   Mounted  in  toto. — 
Approximately, 

48    hours   viewed   from   above   and  below. 

oa  tt  a  "  "  «  " 

30 

24 

18 

12 

64-72  "  "  '•  " 

96  hours  (studied  in  alcohol  under  the  dissecting  microscope). 


a  u  u  <( 

U  U  «  M 

a  u  it  a 


116  Animal  Micrology 

II.   Sections. — 

48  hours,  transverse,  sagittal,  and  frontal. 
36      "                "  " 

30      "  "  " 

24      «  «  « 

18      "  " 

10      "  " 

72      "  "  «  « 

96      "  "  " 

The  number  of  embryos  needed  for  the  above  preparations  is  as  follows: 
5   embryos  of  18  hours   (27-29  somites). 
4        "  "    36      "       (15-18        "      ). 

3  "30      "        (10-14        "      ). 

4  "  "    24      "       (  4-6         "      ). 

2  «  u    18      u 

1  "  «  12      " 

1  "  "  60      " 

3  "  "  64-72  hours  (cervical  flexure  formed). 
3  "  "  96  hours. 

2.  In  Measuring  the  Length  of  Embryos  some  embryologists  (e.  g., 
Minot)  measure  the  greatest  length  of  the  embryo  along  a  straight  line 
(limbs  not  included)  when  the  embryo  is  in  its  normal  attitude;  conse- 
quently in  some  early  stages  where  the  embryo  is  greatly  flexed  the 
neck-bend  would  be  the  point  to  which  to  measure  instead  of  the  tip  of 
the  head,  because  it  is  the  most  anterior  region;  in  stages  where  the 
embryo  is  straight,  the  head  would  be  included.  Other  embryologists 
(e.  g.,  His  and  German  authors  in  general)  make  use  exclusively  of  the 
so-called  "neck-length ;"  that  is,  the  distance  in  a  straight  line  between 
the  neck-bend  and  the  caudal-bend.  In  this  volume  the  full  length 
measurements  are  employed  unless  otherwise  specified. 

3.  To  Mark  Anterior  and  Posterior  Ends  of  Young  Chick  Embryos  in 
blastoderms  which  still  have  a  homogeneous  aspect,  Duval's  osmic  acid 
method  is  very  useful.  With  a  strip  of  paper  5  mm.  wide  by  50  mm. 
long  a  triangular  bottomless  box  with  narrow  base  is  constructed.  This 
is  placed  on  the  yolk  inclosing  the  blastoderm  in  such  a  position  that  the 
base  of  the  triangle  corresponds  to  what  will  be  the  anterior  region  of 
the  embryo  (for  orientation  of  embryo  in  the  egg  see  step  1  of  the  prac- 
tical exercise).  Press  the  box  down  against  the  yolk  and  fill  it  with  a 
0.3  per  cent,  aqueous  solution  of  osmic  acid.  In  a  short  time  the  prepa- 
ration begins  to  darken  and  the  osmic  acid  should  be  removed.  The 
blastoderm  may  then  be  removed  in  the  ordinary  manner  and  fixed  as 
desired  (Duval  used  chromic  acid  for  fixing).    However,  it  is  very  diffi- 


Chapter  XVI:   Some  Embryological  Methods  117 

cult  to  separate  the  blastoderm  from  the  egg  during  the  first  24  hours  of 
incubation  and  it  is  advisable  therefore,  to  fix  and  harden  both  together 
and  to  remove  the  blastoderm  later.*  The  blackened  area  affords  a 
convenient  means  of  orienting  the  preparation  for  sectioning. 

4.  For  the  Embryology  of  Teleosts  the  following  are  the  most  useful 
mounted  stages: 

I.  Whole  mounts. — Two-,  4-,  8-,  16-,  32-,  and  64-cell  stages  (only  the 
blastodisc  segments);  early  periblast;  late  periblast;  early  germ-ring; 
embryonic  shield;  various  stages  of  early  embryos,  such  as  embryos  of 
45,  50  and  60  hours. 

II.  Sections  (paraffin). — Four,  16,  and  32  cells  (vertical  sections  parallel 
to  the  first  plane  of  cleavage);  late  cleavage  (vertical  sections);  early, 
mid,  and  late  periblast  (vertical  sections);  transverse  and  sagittal  sec- 
tions of  early  germ-ring,  embryonic  shield,  early  embryo,  late  germ-ring, 
and  closing  of  blastopore,  respectively. 

All  stages  may  be  fixed  in  picro-acetic  (reagent  23,  Appendix  B)  for 
30  to  40  minutes.  Later  stages  may  also  be  fixed  in  Hermann's  fluid 
(reagent  26)  2  to  4  minutes.  Eggs  fixed  in  Hermann's  fluid  should  be 
washed  for  an  hour  in  frequent  changes  of  water,  and  the  membrane  of 
each  egg  should  then  be  pricked  with  a  needle  before  passing  it  through 
the  series  of  alcohol.  The  eggs  are  finally  preserved  in  83  per  cent, 
alcohol.  (Child  finds  that  fixation  for  about  a  minute  in  10  per  cent, 
acetic  acid  saturated  with  corrosive  sublimate,  followed  by  10  per  cent, 
formalin,  gives  good  results  without  the  yolk  becoming  hard.) 

Before  the  preserved  material  can  be  mounted  in  toto  or  sectioned, 
the  essential  part  (the  blastoderm)  must  be  dissected  off  under  a  dissect- 
ing lens  by  means  of  sharp  needles.  If  the  blastoderms  are  to  be 
mounted  entire  they  may  be  passed  down  through  the  alcohols  (see  Wal- 
ton's device,  memorandum  4,  chap,  iii),  stained  in  Conklin's  hematoxylin 
(reagent  48,  Appendix  B),  then  dehydrated  and  mounted  in  the  usual 
way.  To  avoid  crushing  the  objects,  the  cover-glass  should  be  supported 
by  means  of  bits  of  broken  cover.  Material  which  is  to  be  sectioned 
may  be  stained  in  toto  or  the  sections  may  be  stained  on  the  slide.  In 
the  latter  event,  to  facilitate  orientation,  it  is  necessary  to  tinge  the 
blastoderms  slightly  with  Bordeaux  red  or  some  other  cytoplasmic  stain 
unless  the  fixing  reagent  has  already  done  so.  For  the  same  reason  it  is  best 
to  imbed  the  material  in  a  watch  glass,  arranging  it  near  the  bottom  of  the 
paraffin  mass  so  that  one  can  see  with  a  microscope  how  to  shape  the 

♦Andrews  (Zeituchrift  filr  wissenschaftliche  Mikroskopie,  Vol.  XXI,  1904,  p.  177)  injects 
picro-sulphuric  acid  (1)  between  the  vitelline  membrane  and  the  blastoderm  and  (2) 
between  the  blastoderm  and  the  yolk,  by  means  of  a  pipette  which  has  a  fine  upcurved 
point.  The  blastoderm  may  then  be  readily  freed  from  the  yolk.  This  operation  should 
be  performed  before  the  egg  has  been  subjected  to  the  action  of  any  reagents. 


118  Animal  Micrology 

paraffin  block  in  order  to  cut  sections  in"  the  proper  plane.  The  immer- 
sion in  the  melted  paraffin  should  not  be  longer  than  5  or  10  minutes. 
The  paraffin  is  best  hardened  under  95  per  cent,  alcohol.  The  sections 
may  be  stained  by  any  of  the  hematoxylin  methods;  iron -hematoxylin 
(reagent  51,  Appendix  B)  yields  excellent  results. 

5.  For  the  Embryology  of  the  Frog  and  other  Amphibia  the  following 
are  the  most  useful  stages : 

I.  Surface  views  (from  side  and  from  animal  pole). — Unsegmented 
egg;  2-,  4-,  and  16-cell  stages;  32  becoming  64  cells;  later  cleavage; 
blastopore  forming;  yolk-plug  stage;  early  medullary  folds;  late  medul- 
lary folds;  later  embryo;  embryo  just  before  hatching;  tadpole  shortly 
after  hatching;  later  stages  of  tadpole  for  gross  dissection. 

II.  Sections. — Late  cleavage;  blastopore  forming  (sagittal);  yolk-plug 
stage  (sagittal);  late  medullary  fold  (transverse  and  sagittal);  later 
embryo  (transverse  and  sagittal);  embryo  just  before  hatching  (trans- 
verse and  sagittal);  tadpole  shortly  after  hatching  (sagittal);  head  and 
anterior  part  of  the  tadpole  about  the  time  the  hind  legs  appear 
(sagittal). 

To  study  amphibian  eggs  entire  use  a  hand  lens  or  dissecting  micro- 
scope. Place  the  eggs  on  a  bit  of  absorbent  cotton  under  70  per  cent, 
alcohol  in  salt  cellars.  The  eggs  are  fragile,  consequently  to  manipulate 
them  use  a  soft  hair  pencil  or  a  current  from  a  pipette.  Use  the  same 
egg  for  surface  view  and  for  sectioning  when  possible. 

Amphibian  eggs  may  be  fixed  (in  masses  of  15  or  20)  in  Gilson's 
mercuro- nitric  (reagent  15,  Appendix  B)  or  in  Worcester's  aceto-formol- 
sublimate  mixture  (reagent  196,  Appendix  B).  Chromic  acid  (reagent  10) 
brings  out  surface  views  well  but  the  material  becomes  very  brittle  and 
does  not  take  stains  readily.  If  surface  views  alone  are  desired  formalin- 
preserved  material  will  answer. 

Before  the  egg  can  be  prepared  for  sectioning  the  thick  albuminous 
coat  which  surrounds  it  must  be  removed.  The  fixed  eggs  may  be  shelled 
out  by  means  of  needles.  Whitman  (American  Naturalist,  Vol.  XXII, 
p.  857)  recommends  putting  the  fixed  eggs  into  a  10  per  cent,  solution  of 
sodium  hypochlorite  diluted  with  5  or  6  volumes  of  water  and  leaving 
them  until  they  can  be  shaken  free.  This  requires  only  a  few  minutes. 
Rinse  the  eggs  in  35  per  cent,  alcohol.  It  is  advisable  to  remove  the 
albuminous  coats  before  hardening  in  alcohol. 

Child  (Zeitschrift  fur  wissenschaftliche  Mikroskopie,  Vol.  XVII, 
1900,  p.  205)  states  that  the  albumen  which  surrounds  many  ova  becomes 
transparent  and  dissolves  if  after  fixation  (in  any  way  except  with 
chromic  acid)  the  ova  are  passed  up  through  the  grades  of  alcohol  to 
80  per  cent.,  hardened,  and  then  passed  down  again  through  the  alcohols 
into  water  which  has  been  slightly  acidified  with  any  acid  except  chromic. 


Chapter  XVI:   Some  Embryological  Methods  119 

Amphibian  eggs  are  so  friable  that  they  must  be  sectioned  in 
celloidin  (see,  however,  Johnson's  asphalt-rubber  method,  chap,  v, 
memorandum  9). 

Stain  the  eggs  in  borax-carmine  (reagent  32,  Appendix  B),  then  pass 
them  through  the  alcohols  (35,  50,  70,  95,  100),  leaving  them  half  an 
hour  in  each  (if  necessary  decolorize  in  acidulated  70  per  cent,  alcohol); 
thence  into  ether-alcohol  for  an  hour  or  more,  followed  by  thin  celloidin 
(12  hours)  and  thick  celloidin  (6  hours).  Imbed  in  the  usual  manner, 
but  clear  in  the  block  and  section  as  directed  in  memorandum  10, 
chap.  vii.    Older  embryos  may  be  sectioned  in  paraffin. 

6.  For  Early  Stages  of  the  Mammalian  Embryo  rabbits  are  commonly 
employed  because  they  breed  readily,  especially  in  the  spring  of  the 
year,  and  the  observer  can  note  the  exact  time  when  the  female  is 
covered  if  she  has  been  kept  separate  from  the  buck  until  she  comes 
into  heat.  The  period  of  gestation  is  4  weeks  and  impregnation  takes 
place  again  immediately  after  littering.  The  two  uteri  of  the  rabbit 
diverge  as  two  anterior  horns  from  the  single  median  vagina  and  each  ter- 
minates in  front  in  a  narrow,  coiled  tube,  the  oviduct  or  Fallopian  tubei 
To  obtain  the  early  stages  the  abdomen  is  slit  open  from  pubis  to  ster- 
num, the  intestinal  tract  is  cut  away  or  pushed  to  one  side,  and  each  ute- 
rus and  oviduct  carefully  removed  and  stretched  out  along  a  glass  plate. 
The  segmenting  ova  are  found  in  the  oviduct  up  to  nearly  70  hours  from 
the  time  of  copulation.  After  that  period  of  time  they  must  be  looked 
for  in  the  uterus.  Fecundation  takes  place  about  9  hours  after  coition. 
While  in  the  oviduct,  with  the  aid  of  a  lens  they  may  sometimes  be  seen 
through  its  walls.  A  segmenting  ovum  once  located,  a  transverse  cut  is 
made  to  one  side  of  it  through  the  wall  of  the  oviduct,  and  the  ovum 
which  is  very  small  is  gently  squeezed  out  by  compressing  the  oviduct 
behind  it.  With  a  spear-headed  needle  or  the  point  of  a  scalpel  the 
ovum  is  conveyed  to  the  fixing  fluid.  In  case  segmenting  ova  are  not 
visible  from  the  exterior  of  the  oviduct,  the  latter  must  be  slit  open 
carefully  with  a  pair  of  fine-pointed  scissors,  and  the  eggs  sought  for 
by  means  of  a  lens.  In  case  no  red  corpora  lutea  are  visible  on  the 
surface  of  the  ovary,  indicating  a  recent  discharge  of  ova  from  the 
Graafian  follicles,  further  search  is  useless. 

Rabbit  ova  of  18  hours  show  4  blastomeres;  36  to 48  hours,  advanced 
segmentation;  72  hours  (about  0.6  mm.  in  diameter;  in  anterior  end  of 
uterus)  show  the  fully  segmented  ovum — an  outer  layer  of  clear,  cubical 
cells,  an  inner  mass  of  irregular  granular  cells;  72  to  90  hours  show 
enlarged  blastodermic  vesicle  and  establishment  of  embryonic  area; 
fifth  and  sixth  days  (0.8  to  4  mm.)  show  germinal  layers;  seventh  day, 
primitive  streak;  8th  day,  medullary  folds. 

The  earlier  stages  (up  to  70  hours)  may  be  fixed  for  from  5  to  8  min 


120  Animal  Micrology 

utes  in  a  0.3  per  cent,  aqueous  solution  of  osmic  acid,  stained  in  picro- 
carmine,  and  transferred  to  a  mixture  of  glycerin  and  water,  equal  parts. 
They  should  remain  in  this  fluid  for  a  week  under  a  bell-jar  so  that  the 
water  gradually  evaporates.  The  object  may  then  be  mounted  in 
formic -glycerin  (formic  acid  1  part,  glycerin  99  parts).  To  avoid  pressure 
of  the  cover-glass,  the  object  should  be  mounted  in  a  cell  or  between  two 
slips  of  paper  or  pieces  of  cover-glass.  If  the  preparation  is  to  be  per- 
manent the  cover-glass  should  be  sealed  (see  chap,  xiii,  II,  A,  6). 

To  render  the  cell  outlines  distinct  stages  of  from  70  to  80  hours  are 
best  treated,  after  rinsing  in  distilled  water,  with  a  1  per  cent,  aqueous 
solution  of  silver  nitrate  for  3  minutes  and  then  exposed  to  light  in  a 
dish  of  distilled  water  until  they  become  brown.  They  are  then  treated 
with  water  and  glycerin  and  mounted  in  formic-glycerin  as  in  the  case  of 
younger  stages. 

For  sections,  the  embryos  should  be  placed  in  Hermann's  (reagent  26, 
Appendix  B)  or  Zenker's  (reagent  6)  fluid  for  about  an  hour,  then  washed 
in  the  customary  way  for  these  methods,  stained  in  borax-carmine  or 
alum -cochineal,  and  sectioned  in  paraffin. 

In  opening  the  uterus,  the  incision  should  always  be  made  along  the 
middle  of  the  free  side,  opposite  the  insertion  of  the  peritoneal  fold, 
because  this  line  of  insertion  marks  the  region  of  attachment  of  the 
embryo  within  the  oviduct.  By  the  seventh  or  eighth  day  the  developing 
ova  have  taken  up  positions  at  intervals  along  the  inner  walls  of  the 
uterus  and  have  become  so  firmly  attached  to  the  mucous  membrane 
that  they  can  no  longer  be  detached  unmutilated.  For  further  particu- 
lars regarding  the  embryology  of  the  rabbit,  the  reader  is  referred  to 
E.  Van  Beneden  and  Charles  Julin's  "  Recherches  sur  la  formation  des 
annexes  foetalis  chez  les  mammiferes,"  Archives  de  Biologie,  Vol.  V 
(1884),  p.  378. 

7.  For  Older  Stages  of  the  Mammalian  Embryo,  pig  embryos  are  com- 
monly employed.  They  may  often  be  procured  in  large  numbers  and 
with  little  trouble  at  the  larger  pork-packing  establishments.  The  most 
valuable  stage  for  study  is  an  embryo  of  from  10  to  13  mm.  in  length. 
In  most  laboratories  it  is  customary  to  make  a  detailed  study  of  an 
embryo  of  about  this  stage  and  then  a  more  general  survey  of  both 
smaller  and  larger  sizes. 

Early  stages  are  much  more  difficult  to  obtain  than  advanced  stages. 
Embryos  of  6  mm.  length  and  over  may  usually  be  readily  located  by 
the  enlargements  which  they  cause  in  the  uterine  walls.  The  uterus 
should  be  handled  carefully  and  opened  as  soon  as  possible.  The 
embryo  is  best  removed  by  means  of  fine  forceps  and  a  horn  spoon.  It  is 
very  delicate  and  should  not  be  handled  roughly.    The  chances  are  that 


Chapter  XVI:   Some  Embryological  Methods  121 

in  removing  the  embryo  the  membranes  will  be  ruptured  and  the  amni- 
otic and  allantoic  fluids  will  escape. 

Submerge  the  embryo  without  removing  the  membranes  in  a  bounti- 
ful supply  of  Kleinenberg's  picro-sulphuric  acid  (reagent  25,  Appendix 
B),  moving  it  about  gently  to  rinse  off  any  coagulum  that  may  form  on 
the  surface. 

Leave  embryos  of  6  to  9  mm.,  2J  hours;  12  to  15  mm.,  4  hours;  20  to 
25  mm.,  6  to  8  hours. 

For  washing  and  subsequent  treatment  see  reagent  25,  Appendix  B. 
Embryos  may  be  stained  in  toto  in  alum -cochineal  (reagent  27)  or  borax- 
carmine  (reagent  32). 

For  studying  the  uterus,  placentation  (diffuse  in  the  pig),  and  embry- 
onic membranes  in  place,  formalin-hardened  material  may  be  used  after 
first  thoroughly  washing  it  in  water. 

For  gross  dissection  of  embryos,  the  specimen  should  be  studied  in 
alcohol  under  the  dissecting  microscope. 

Because  of  the  asymmetry  of  young  embryos  it  is  impossible  to  secure 
strictly  transverse,  sagittal,  and  frontal  sections.  Minot  recommends, 
therefore,  that  for  practical  purposes  the  plane  of  section  be  taken  with 
regard  to  the  head  alone  irrespective  of  how  it  may  cut  the  other  parts  of 
the  body  and  suggests  the  floor  of  the  fourth  ventricle  of  the  brain  as  the 
guide  for  orientation.  In  his  Laboratory  Text-Book  of  Embryology  he 
especially  recommends  that  each  student  prepare  sections  of  the  follow- 
ing stages  of  pig  embryos:  9  mm.,  transverse  and  sagittal,  frontal  of  the 
head;  6  mm.,  transverse,  frontal  of  the  head;  17  mm.,  transverse  and 
sagittal,  frontal  of  the  head;  20  mm.,  transverse  and  sagittal,  frontal  of 
the  head;  24  mm.,  frontal  of  the  head. 

8.  For  the  Stages  of  Maturation,  Fertilization,  and  Segmentation  in  Mam- 
mals white  mice  will  prove  most  useful  because  these  processes  are  better 
known  in  them  than  in  other  mammals;  furthermore,  an  abundance  of 
material  may  be  procured.  The  ovum,  however,  is  extremely  small, 
measuring  only  59  microns  in  diameter.  It  is  surrounded  by  a  very  thin 
zona  pellucida  (1.2  microns).  In  the  majority  of  ova  (80  to  90  per  cent.) 
only  a  single  polar  body  is  formed;  it  corresponds  in  every  way  appar- 
ently to  the  second  polar  body  when  two  are  formed.  It  has  been 
inferred,  therefore,  that  probably  in  the  mouse  the  first  polar  body  ordi- 
narily forms  at  an  early  stage  in  the  history  of  the  germ  cells  and  is  thus 
overlooked  by  observers. 

When  two  polar  bodies  have  been  observed,  the  first  always  appears 
while  the  egg  is  yet  in  the  Graafian  follicle  of  the  ovary;  the  second,  after 
the  egg  has  entered  the  Fallopian  tube.  When  only  one  polar  body  is 
present  it,  likewise,  appears  after  the  egg  has  entered  the  tube.    The 


122  Animal  Micrology 

spermatozoon  reaches  the  egg  in  the  upper  end  of  the  Fallopian  tube 
some  time  after  coitus.  Formation  of  the  single  (or  the  second)  polar 
body  and  the  entrance  of  the  spermatozoon  may  be  found  in  the  same  egg. 

The  female  comes  into  heat  21  days  after  littering  and  coitus  can 
take  place  only  when  she  is  in  heat  because  at  other  times  the  vagina  is 
closed.  During  heat  the  periovarial  space  and  the  beginning  of  the  Fal- 
lopian tube  are  distended  with  a  clear  fluid.  Ovulation  occurs  before  or 
at  the  time  of  coitus.  (See  "  Die  Befruchtung  und  Furchung  des  Eies 
der  Maus"  by  Sobotta:  Archiv  fur  mikroskopische  Anatomie,  Vol.  XLV 
[1895],  pp.  15-93,  Plates  II-IV.) 

9.  Artificial  Fecundation  when  it  can  be  practiced  is  the  most  convenient 
means  of  securing  early  stages  of  development.  This  is  possible  with 
many  worms,  coelenterates,  echinoderms,  cyclostomes,  teleosts,  and  anu- 
ran  amphibia. 

a)  In  echinoderms  (e.  g.,  sea  urchin)  the  female  is  cut  open  and  a  num- 
ber of  the  living  eggs  transferred  to  a  watch-glass  which  contains  fresh 
sea  water.  The  testes  of  a  male  are  teased  out  in  sea  water  and  a  drop 
of  the  mixture  is  conveyed  by  means  of  a  pipette  into  the  dish  containing 
eggs.  Immediately  upon  fertilization  a  membrane  forms  around  each 
fertilized  egg.  In  about  40  to  50  minutes  after  fertilization  the  signs  of 
the  first  cleavage  should  appear.  The  blastula  forms  in  about  6  hours, 
and  the  gastrula  in  about  12  hours.  For  the  study  of  fertilization,  etc., 
the  following  stages  should  be  fixed  in  picro-acetic  (reagent  23,  Appen- 
dix B)  for  30  minutes  and  stained  in  Conklin's  hematoxylin  (reagent  48); 
5  minutes  after  fertilization,  nucleus  giving  off  polar  bodies;  30  minutes 
after  fertilization,  approaching  pronuclei ;  50  to  55  minutes  after  fertiliza- 
tion, division  of  nucleus  (mitotic  figure)  in  the  first  cleavage. 

b)  In  amphibia  (e.  g.,  frog)  both  male  and  female  are  cut  open,  the 
vasa  deferentia  or  testes  are  teased  out  in  a  watch-glass  full  of  water  and 
the  ova  are  then  removed  from  the  lower  ends  of  the  oviducts  and  placed 
in  this  water.  After  fertilization  the  eggs  should  be  placed  in  glass 
dishes  in  not  over  4  inches  of  water.  Many  eggs  should  not  be  placed 
in  one  dish.     See  also  memorandum  5. 

c)  In  teleosts  the  eggs  are  obtained  by  stripping  the  female  when  she 
is  in  spawning  condition.  At  such  times  the  eggs  are  loose  in  the  body 
cavity  and  may  be  pressed  out  by  gently  manipulating  the  belly  of  the 
fish.  The  head  of  the  fish  should  be  held  in  one  hand,  the  tail  in  the 
other  and  the  thumb  or  the  thumb  and  forefinger  used  to  press  out  the 
ova;  the  vent  of  the  fish  should  remain  submerged  in  water.  The  milt 
of  the  male  is  obtained  in  the  same  manner  and  in  the  same  dish.  Eggs 
and  sperm  are  then  gently  stirred  about  by  means  of  a  feather  to  insure 
thorough  mixing.  However,  in  some  teleosts  (e.  g.,  stickleback)  it  is 
necessary  to  kill  the  male  and  tease  out  the  testes.     In  the  cunner  (Cten- 


Chapter  XVI:   Some  Embryological  Methods  123 

olabrus)  10  minutes  after  fertilization  the  formation  of  blastodisc  and 
polar  bodies  may  be  observed;  30  to  33  minutes  after  fertilization  the 
two  pronuclei  may  be  found  in  close  approximation. 

If  other  than  the  very  early  stages  are  required,  the  fertilized  eggs 
must  be  transferred  to  a  hatching-box.  This  is  best  done  by  means  of  a 
horn  spoon  and  a  feather.  The  hatching-box  must  be  provided  with  a 
very  gentle  (drop  by  drop)  stream  of  running  water.  According  to  Exner 
the  eggs  are  best  placed  in  the  box  on  a  layer  of  glass  rods  which  are 
from  one-twelfth  to  one-sixth  of  an  inch  apart.  Three-eighths  of  an 
inch  below  the  rods  there  should  be  a  layer  of  pebbles  covering  the  floor 
of  the  box.  Dead  eggs,  recognizable  by  their  opacity,  should  be  removed 
at  least  once  a  day.     See  also  memorandum  4. 

10.  For  the  Study  of  Early  Cleavage  in  Living  Material  the  eggs  of  some 
of  the  water  snails  afford  an  abundance  of  excellent  material.  By  watch- 
ing aquaria  which  contain  snails  the  fresh  material  can  easily  be  obtained 
during  the  spring  and  summer.  Twigs  and  bits  of  board  to  which  the 
egg-masses  may  be  attached  should  be  placed  in  the  aquaria. 

11.  For  the  Study  of  the  Formation  of  Polar  Bodies,  Fertilization,  and  Early 
Cleavage  in  Sections  nothing  surpasses  the  eggs  of  Ascaris.  The  Ascaris 
(A.  megalocephala)  from  the  horse  is  preferable  although  A.  lumbri- 
coides  from  the  pig  will  answer. 

The  ovisacs,  two  in  number,  are  very  long  convoluted  tubes.  Differ- 
ent regions  contain  eggs  in  different  stages  of  development.  The 
thicker  tubes  toward  the  anterior  end  of  the  animal  contain  cleavage 
stages;  back  of  these  are  cells  showing  extrusion  of  the  polar  bodies  and 
fertilization  stages.  The  material  must  be  fresh;  either  bring  the  live 
Ascaris  to  the  laboratory  or  take  the  fixing  fluid  to  the  place  for  obtain- 
ing the  material.  Slit  open  the  abdominal  wall  of  the  worm  and  remove 
the  ovisacs  and  after  separating  the  numerous  convolutions  somewhat, 
fix  them  entire  for  24  hours  in  picro-acetic  acid  (reagent  23,  Appendix 
B),  or  for  15  to  25  minutes  in  acetic-alcohol-chloroform  (reagent  26)  satu- 
rated with  corrosive  sublimate.  Preserve  in  80  per  cent,  alcohol.  To 
locate  eggs  of  the  desired  stage  tease  out  eggs  at  intervals  along  the 
ovisacs,  stain  with  acid  carmine  (reagent  37)  and  examine.  The  proper 
region  once  located,  cut  out  small  lengths  of  the  tube,  imbed  it  in  par- 
affin and  make  thin  transverse  sections.  In  order  to  keep  the  eggs  from 
shriveling,  the  bath  in  hot  paraffin  must  be  curtailed.  Use  the  method 
for  delicate  objects  (chap,  vi,  vii).  Stain  by  the  iron-hematoxylin  method 
(reagent  51,  Appendix  B). 

12.  Directions  for  Orienting  Serial  Sections. — a)  In  mounting  transverse 
sections  (sections  across  the  main  axis  of  the  object),  the  sections,  begin- 
ning at  the  anterior  end  of  the  object,  are  laid  on  the  slide  in  the  same 
sequence  as  the  reading  on  the  page  of  a  book.     In  order  to  have  right 


124:  Animal  Micrology 

and  left  sides  and  dorsal  and  ventral  surfaces  in  proper  relation  to  the 
observer,  mount  the  object  in  such  a  way  that  in  cutting  the  knife  will 
enter  it  on  the  right  side  and  at  the  anterior  end.  In  mounting,  each 
section  (or  strip  of  ribbon)  is  turned  over  and  mounted  with  its  posterior 
face  toward  the  observer  and  its  ventral  edge  toward  the  upper  edge  of 
the  slide.  Leave  room  at  one  end  of  the  slide  (see  chap,  vi,  I,  10)  for  a 
label  and  also  a  small  margin  at  the  opposite  side. 

b)  To  get  proper  orientation  of  frontal  sections  (sections  lengthwise 
of  the  object  in  a  plane  including  right  and  left  sides)  arrange  the  object 
so  that  the  knife  will  enter  it  on  the  right  side  and  slice  off  the  dorsal 
surface  first.  Mount  sections,  with  their  posterior  ends  toward  the 
upper  edge  of  the  slide  placing  the  first  section  of  the  series  to  the  left 
end  of  the  upper  row.  This  throws  left  and  right,  dorsal  and  ventral 
into  their  proper  position  as  viewed  through  the  compound  microscope 
and  the  observer  looks  from  the  dorsal  toward  the  ventral  aspect  of 
the  object. 

c)  To  mount  sagittal  sections  (sections  lengthwise  of  the  object  in  a 
plane  including  ventral  and  dorsal  sides)  arrange  the  object  in  such  a 
position  that  the  knife  enters  the  ventral  surface  and  slices  off  the  left 
side  first.  Turn  the  section  (or  ribbon)  over  and  mount  with  the  pos- 
terior end  toward  the  upper  edge  of  the  slide,  placing  the  first  section 
of  the  series  at  the  left  end  of  the  upper  row.  Through  the  compound 
microscope,  the  observer  views  the  object  from  the  right  toward  the  left. 
The  head  will  appear  to  be  toward  the  upper  end  of  the  slide,  the  dorsal 
surface  toward  the  left. 

It  is  frequently  advantageous  to  have  the  imbedding  mass  trimmed 
unsymmetrically  by  leaving  the  edge  which  first  comes  in  contact  with 
the  knife  longer  than  the  opposite  edge.  One  may  thus  readily  discover 
if  a  section  or  part  of  a  series  has  been  accidentally  turned  over. 

13.  Orientation  of  Objects  in  the  Imbedding  Mass  so  that  sections  can 
be  cut  accurately  in  definite  planes  is  frequently  difficult  to  accomplish. 
The  following  methods  are  useful  in  many  instances: 

I.  For  paraffin  sections. — With  a  soft  pencil  rule  the  strip  of 
paper  which  is  to  be  used  for  making  the  imbedding-box  into  small 
squares  or  rectangles.  After  imbedding,  upon  removal  of  the  paper  a 
copy  of  the  pencil  marks  will  be  found  upon  the  block  of  paraffin.  If 
the  object  has  been  arranged  in  the  melted  paraffin  with  reference  to 
these  lines,  it  is  easy  so  to  arrange  the  block  in  the  microtome  as  to  cut 
the  object  along  any  desired  plane.  It  is  frequently  an  aid  to  orientation 
by  this  method  to  have  one  of  the  central  ruled  lines  broader  than  the 
others,  or  double. 

Small  objects  which  cannot  conveniently  be  oriented  in  melted  par- 
affin may  be  properly  oriented  and  fixed  to  a  small  strip  of  paper  ruled 


Chapter  XVI :   Some  Embryological  Methods  125 

as  above,  before  they  are  placed  in  the  paraffin  bath,  by  a  mixture  of 
clove  oil  and  collodion  of  about  the  consistency  of  thick  molasses,  as  in 
Patton's  method  (Zeitschrift  fur  wissenschaftliche  Mikroskopie,  Vol.  XI 
[1894],  p.  13).  One  or  a  number  of  small  objects  which  have  previously 
been  cleared  in  oil  of  bergamot  or  cloves  are  mounted  in  small  separate 
droplets  of  the  reagent  and  oriented  under  a  dissecting  lens  with  refer- 
ence to  the  ruled  lines.  The  paper  is  then  placed  in  turpentine  which 
washes  out  the  clove  oil  and  fixes  the  object  in  place.  The  paper  with 
objects  attached  is  then  passed  through  melted  paraffin  and  imbedded  in 
the  ordinary  way.  Upon  removal  of  the  paper  from  the  hardened  block 
a  sufficient  number  of  pencil  marks  remain  to  be  used  as  a  guide  in  sec- 
tioning.   Instead  of  pencil  marks  Patton  employed  ribbed  paper. 

II.     For  celloidin  sections. — 

Eycleshymer's  Methods. — a.  For  imbedding,  metal  boxes  made  of  two 
|_s  (Fig.  30)  are  used.  The  Ls  are  held  together  by  overlapping  strips. 
The  ends  and  sides  of  the  box  are  perforated  at  regular  intervals  by 
small  holes  which  have  been  drilled  opposite  one  another  in  such  a  way 
that  threads  drawn  through  them  are  parallel.  Threads  of  silk  are  run 
through  the  holes  from  side  to  side,  drawn  taut,  and  cemented  to  the 
outside  of  the  box  with  a  drop  of  celloidin.  Each  piece  of  thread  should 
have  an  end  two  or  three  inches  long  hanging  outside  the  box.  A  piece 
of  heavy  blotting  paper  is  used  as  a  bottom  for  the  box.  The  object  is 
oriented  on  the  parallel  threads  and  the  imbedding  mass  poured  in  and 
hardened.  The  loose  ends  of  the  threads  are  then  soaked  in  a  solution 
of  thin  celloidin  which  contains  lamp-black,  the  celloidin  drops  holding 
the  threads  taut  are  dissolved  by  a  drop  of  ether-alcohol,  and  the  black- 
ened ends  are  drawn  through  the  block  of  celloidin.  The  lamp-black 
leaves  distinct  black  lines  through  the  mass  which  will  serve  for  properly 
orienting  the  celloidin  block  on  the  microtome. 

This  method  is  valuable  also  in  reconstructions  from  sections  (see 
chap.  xvii).  In  such  work  it  is  very  desirable  to  establish  "reconstruc- 
tion points "  to  guide  in  fitting  the  wax  plates  together  properly.  The 
black  rings  of  lamp-black  left  in  the  sections  answer  admirably  for  this 
purpose. 

b.  For  small  objects  in  which  reconstruction  points  are  not  required 
Eycleshymer  uses  fine  insect  pins  from  which  the  heads  have  been 
clipped  and  the  headless  ends  loosely  inserted  in  handles.  The  objects 
are  mounted  on  the  points  of  the  pins  and  oriented  in  the  desired  posi- 
tion. Each  pin  is  then  removed  from  its  handle,  and  the  free  end  is 
inserted  from  below  into  a  small  perforation  which  has  been  made  by 
passing  a  somewhat  larger  pin  lengthwise  through  a  cork.  A  number 
of  pins  may  be  mounted  on  the  same  cork.  To  prevent  the  objects  from 
becoming  dry  the  cork  must  frequently  be  inserted  into  the  mouth  of  a 


126  Animal  Micrology 

vial  full  of  alcohol  in  such  a  way  that  the  objects  are  immersed.  If 
desired,  the  objects  may  be  sketched  in  situ  under  alcohol  by  weighting 
the  cork  with  lead  and  placing  it  in  a  beaker  of  alcohol.  To  pass  the 
objects  through  the  various  grades  of  alcohol,  etc.,  simply  transfer  the 
cork  bearing  them  to  successive  vials  of  proper  size  containing  the  dif- 
ferent fluids.  For  imbedding  in  celloidin  use  the  method  given  on  p.  59, 
steps  2  ff .  When  the  celloidin  mass  has  hardened  the  paper  is  removed 
and  the  pins  are  drawn  out  through  the  cork,  thus  leaving  the  objects  in 
place  ready  for  sectioning. 

14.  Human  Embryos  of  all  ages  are  very  valuable  material  for  scien- 
tific purposes.  Physicians  and  surgeons  are  urged  to  preserve  such  mate- 
rial properly  and  turn  it  over  to  some  competent  embryologist.  Very 
young  human  embryos  are  exceedingly  desirable. 

An  excellent  fixing  reagent,  the  ingredients  of  which  a  physician  can 
usually  readily  procure,  is  the  acetic-alcohol-chloroform  mixture  described 
in  Appendix  B,  reagent  26.  The  embryo  should  remain  in  this  fluid 
from  6  to  24  hours  according  to  size  and  then  be  preserved  in  80  per  cent, 
alcohol  (or  commercial  alcohol  to  which  has  been  added  about  one-fifth 
its  volume  of  distilled  water).  Use  a  wide-mouthed  bottle  with  tightly 
fitting  stopper. 

Zenker's  fluid  (reagent  6,  Appendix  B)  is  better  for  larger-sized 
embryos.  Material  should  be  left  in  it  from  18  hours  to  several  days. 
For  washing  and  preserving  follow  the  directions  given  under  the 
description  of  the  fluid.  For  fetuses  use  a  fruit  jar  of  such  a  size  that  the 
embryo  can  be  kept  in  about  10  times  its  volume  of  fluid. 

In  case  the  above  fluids  are  not  available,  the  material  may  be  placed 
in  10  per  cent,  formalin  (1  part  of  commercial  formalin  to  9  parts  of  dis- 
tilled water)  and  left  indefinitely.  As  a  last  resort,  if  no  other  fixing 
reagent  is  available,  the  embryo  may  be  placed  in  the  strongest  alcohol 
which  can  be  secured  and  later  transferred  to  80  per  cent,  alcohol  for 
preservation. 

The  specimen  should  not  be  handled  nor  allowed  to  lie  in  water. 
When  the  proper  reagents  are  not  at  hand,  carefully  wrap  the  object  in 
cloth  and  keep  it  on  ice  if  possible  until  they  can  be  secured.  Very 
small  embryos  may  be  fixed  and  preserved  with  membranes  intact;  older 
ones  (6  weeks  to  3  months)  should  have  the  membranes  ruptured.  To 
secure  the  best  fixation  of  fetuses  (2  months  and  beyond),  the  specimen 
should  be  divided,  or  at  least  the  body  cavity  should  be  opened. 


CHAPTER  XVII 

RECONSTRUCTION  OF  OBJECTS  FROM  SECTIONS 

In  investigating  objects  which  possess  complex  internal  cavities 
or  complicated  structures,  it  is  frequently  very  difficult  to  gain  an 
adequate  idea  from  the  direct  study  of  serial  sections,  or  by  means 
of  macerated  or  teased  preparations,  consequently,  various  methods 
of  plastic  or  geometrical  reconstructions  from  the  sections  are 
resorted  to.  For  such  reconstruction,  sections  must  be  of  uniform 
thickness,  serial,  and  they  must  possess  similar  orientation. 

RECONSTRUCTION  IN  WAX 

Born's  method  of  constructing  wax  models  of  objects  from 
serial  sections  is  widely  used  for  both  embryological  and  anatomical 
subjects.  The  thickness  of  the  sections,  the  magnification  of  the 
microscope,  and  the  plane  of  section  must  be  known. 

Wax  plates  are  prepared  as  many  times  thicker  than  the  actual 
sections  as  the  latter  will  be  magnified  in  diameters.  For  example, 
if  the  serial  sections  are  ^  of  a  millimeter  thick  (33^  microns), 
and  they  are  to  be  magnified  60  diameters,  then  the  wax  plates 
must  be  made  60  times  as  thick  as  the  sections,  or  2  millimeters 
thick.  This  is  the  thickness  commonly  used.  Count  the  number 
of  sections  to  be  reconstructed,  and  prepare  an  equal  number  of 
plates. 

Preparation  of  the  Wax  Plates 
(a)  The  original  method. — 1.  To  prepare  wax  plates  of  the  proper 
thickness  (2  mm.)  use  several  straight-walled  rectangular  tin  pans  25  mm. 
deep  and  measuring  270x230  mm.  in  area. 

2.  In  order  to  make  the  beeswax  which  is  to  be  used  flexible,  add  a 
little  turpentine  to  it.  The  specific  gravity  of  the  mixture  will  be  about 
0.95,  and  the  weight  necessary  to  make  a  plate  of  wax  2  mm.  thick  in  one 
of  the  pans  will  be  about  118  grams. 

3.  Fill  one  of  the  pans  with  boiling  water  to  the  depth  of  about 
1.5  cm.,  melt  118  grams  of  the  prepared  wax  and  pour  it  upon  the  water. 
The  wax  should  spread  evenly  over  the  surface  of  the  water  if  both  wax 
and  water  are  sufficiently  hot.     If  gaps  remain,  close  them  by  drawing  a 

127 


128  Animal  Micrology 

glass  slide  over  the  surface  of  the  wax.  To  prevent  the  plate  from 
splitting  while  cooling,  after  it  has  stiffened  somewhat,  cut  the  edges 
free  from  the  walls  of  the  pan.  When  the  water  has  become  tepid, 
remove  the  wax  plate  to  a  flat  support  and  leave  it  to  harden. 

(b)  Huber's  method. — Several  instruments  have  been  devised  for 
making  the  plates  more  rapidly  and  more  accurately  than  by  the  original 
method.  Huber's  apparatus,  for  instance,  consists  of  a  heavy  cast-iron 
plate  with  moveable  side  pieces  which  can  be  adjusted  to  a  height  cor- 
responding to  the  desired  thickness  of  the  wax  plates.  The  whole 
instrument  is  supported  upon  three  adjustable  legs,  by  means  of  which 
it  can  be  made  exactly  level.  Melted  beeswax  slightly  in  excess  of  the 
quantity  necessary  for  a  wax  plate  is  poured  on  to  the  iron  plate  in  an 
even  layer,  and  rolled  out  with  a  hot  roller  until  the  roller  comes  to  run 
directly  on  the  side  pieces  of  the  instrument.  When  the  wax  plate  is 
cool  enough  to  handle  it  may  be  placed  in  a  pan  of  cold  water  to  harden. 

Practical  Exercise. — When  possible  an  outline  drawing  of  the 
part  to  be  reconstructed  should  be  made  before  it  is  sectioned. 

1.  Reconstruct  the  heart  of  a  chick  at  the  end  of  the  third 
day  of  incubation,  under  a  magnification  of  60  diameters.  For 
this  magnification,  if  it  is  desired  to  use  a  wax  plate  2  mm.  thick, 
the  original  sections  should  have  been  33.3  microns  thick. 

2.  Place  a  sheet  of  blue  tracing-paper  on  the  wax  plate  with 
the  colored  side  toward  it.  Over  the  tracing-paper  place  a  sheet 
of  ordinary  drawing-paper.  With  the  aid  of  a  camera  lucida  or 
other  projection  apparatus,  outline  on  the  drawing-paper  the  part 
to  be  reconstructed.  In  doing  this  the  outline  is  also  traced  in 
blue  on  the  wax.  Number  each  drawing,  and  also  indicate  the 
number  of  the  section  on  the  slide  to  which  it  corresponds;  also 
number  the  wax  plates  with  reference  to  the  drawings. 

3.  Lay  the  wax  plate  on  a  suitable  flat  surface,  and  cut  out  the 
outlined  parts  with  a  sharp,  narrow-bladed  knife.  Leave  bridges 
of  wax  to  hold  in  place  the  parts  that  would  otherwise  be  separate 
pieces.  Pile  up  the  successive  sections  in  proper  sequence  as 
they  are  cut  out. 

4.  In  finally  putting  the  model  together,  accurately  adjust  the 
parts  (for  reconstruction  points  see  chap,  xvi,  memorandum  13, 
II a),  and  build  up  the  model  in  blocks  of  five  sections  each 
(Bardeen's  suggestion).      If  necessary,  unite  the  essential  parts 


Chapter  XVII:  Reconstruction  of  Objects  from  Sections     129 

by  means   of   pins   or   fine   nails.      Remove   all    temporary   wax 
bridges  (see  3)  by  means  of  a  hot  knife. 

When  all  blocks  are  properly  adjusted  and  united,  smoothe 
over  the  surface  by  means  of  a  hot  spatula. 

MEMORANDA 

1.  Geometrical  Reconstructions,  first  described  by  Professor  His,  are 
often  all  that  is  necessary  to  give  one  the  desired  information  about 
internal  organs.  Before  sectioning,  an  outline  drawing  of  the  object  is 
made  in  a  plane  at  right  angles  to  the  intended  plane  of  section,  and 
under  the  same  magnification  that  will  be  used  for  the  reconstructed 
drawing.  For  example,  if  the  sections  are  to  be  transverse,  the  outline 
drawing  of  the  object  would  be  a  profile  view  from  the  side.  After 
sectioning  the  object,  each  section  is  drawn  under  the  same  magnification 
as  was  used  for  the  outline  drawing. 

To  reconstruct  any  special  part  of  the  object,  draw  a  median  line  on 
the  outline  drawing  corresponding  to  the  long  axis  of*  the  object.  At 
right  angles  to  this  line,  draw  a  series  of  equidistant  parallel  lines 
corresponding  in  positions  to  the  sections  that  have  been  made.  For 
example,  if  the  magnification  is  100  diameters  and  the  sections  10  microns 
thick,  then  the  parallel  lines  must  be  1  mm.  apart.  Then,  beginning 
with  the  first  section,  indicate  by  dots  in  the  proper  plane  in  the  profile 
drawing  the  relative  distances  of  the  part  in  the  sections  above  or  below 
the  median  line  along  the  proper  one  of  the  parallel  lines.  All  of  the 
sections  having  thus  been  plotted,  connect  the  dots  of  corresponding 
parts  in  the  successive  zones.  It  is  frequently  sufficient  to  reconstruct 
only  every  fifth  or  even  every  tenth  section.  When  the  plane  of  section 
is  not  quite  at  right  angles  to  the  axis  of  the  object,  an  equal  alteration 
of  angle  must  be  made  between  the  median  line  of  the  outline  drawing 
and  the  parallel  lines. 

Such  a  reconstruction  as  the  above  would  give  lateral  views  of  the 
various  internal  parts.  To  get  their  aspects  as  seen  from  above  or 
below,  the  original  outline  drawing  of  the  specimen  as  a  whole  should 
have  been  made  from  this  point  of  view  instead  of  from  the  side.  In 
actual  work  one  should  make  reconstructions  in  both  planes. 

2.  A  Special  Drawing-Table  for  rapid  and  convenient  drawing  of  sections 
for  reconstruction  has  been  devised  by  Bardeen.  For  details,  see  Johns 
Hopkins  Bulletin,  XII,  p.  148. 


APPENDICES 


APPENDIX  A 

THE  MICROSCOPE  AND  ITS  OPTICAL  PRINCIPLES 

For  an  understanding  of  the  optical  principles  involved  in 
microscopy,  four  things  must  be  borne  in  mind  with  regard  to  a 
ray  of  ordinary  daylight: 

1.  It  has  an  appreciable  breadth. 

2.  It  travels  in  a  straight  line  in  a  homogeneous  medium. 

3.  It  is  bent  {refracted)  in  passing  obliquely  from  one  medium 
into  another  of  different  density. 

4.  It  is  in  reality  a  composite  of  a  number  of  different  colored 
rays,  ranging  from  violet  to  red,  and  each  of  these  has  a  different 
refrangibility. 

The  amount  of  refraction  undergone  by  light  in  a  given  case 
depends  upon  the  difference  in  density  of  the  two  media  which 
the  light  traverses.  Thus,  glass  is  denser  than 
air,  hence,  in  passing  from  air  obliquely  through 
a  glass  plate  (Fig.  41),  a  ray  of  light  A B  would 
be  bent  out  of  its  original  course.  On  reaching 
the  air  again,  however,  it  would  resume  its 
original  direction,  although  it  would  be  dis- 
placed to  an  amount  equal  to  the  distance  ., 
between  A  and  A ' .  It  is  on  account  of  such 
displacement  that  an  object  in  water,  for  ex- 
ample, appears  to  be  at  a  different  point  from  where  it  really  is. 
On  the  other  hand,  after  traversing  a  prism,  a  ray  does  not 
resume  its  former  direction,  but  takes  a  new  course  upon  leav- 
ing as  well  as  upon  entering  the  prism  (Fig. 
42).  This  new  direction  is  always  toward 
the  base  of  the  prism,  and  the  amount  of  de- 
viation depends  upon  the  shape  and  density 
of  the  prism.  If  the  base  is  down,  then  the 
Fig.  42.  ray  is  bent  downward ;  if  the  apex  is  down, 

the  ray  still  deviates  towards  the  base,  that  is,  it  is  bent  upward 

133 


Fig.  41. 


134  Animal  Micrology 

Lenses. — Each  of  the  two  principal  forms  of  lenses  is  in  effect 
practically  two  prisms,  (1)  with  the  bases  placed  together 
(Fig.  43  a,  convex  lens),  cr,  (2)  with  the  apices  together  (Fig.  436, 
concave  lens) . 

In  the  convex  lens,  since  rays  of  light  are  refracted  toward  the 
bases  of  the  respective  prisms,  they  will  converge ;  in  the  concave 
lens,  for  the  same  reason,  they  will  diverge.  The  terms  conver- 
ging lens  and  diverging  lens,  therefore,  are  used  frequently  as 
synonymous  with  the  terms  convex  lens  and  concave  lens.  All 
lenses  are  modifications  or  combinations  of  these  two  types. 


a  b  \ci 

Fig.  43.  Fig.  44. 

If  parallel  rays  of  light  pass  through  a  convex  lens  (Fig.  44) 
they  are  so  refracted  as  to  meet  in  one  point  F,  which  is  termed, 
in  consequence,  the  focal  point  or  principal  focus.  If,  on  the 
other  hand,  the  source  of  light  be  placed  at  the  focal  point,  then, 
after  traversing  the  lens,  the  rays  of  light  will  emerge  parallel. 
If  parallel  rays  of  light  came  from  the  opposite  side  of  the  lens, 
manifestly  there  would  be  a  second  focal  point  at  F' .  The  two 
principal  foci  are  termed  conjugate  foci,  and  will  be  equidistant 
from  the  center  of  the  lens  when  both  sides  of  the  lens  have  equal 
curvature. 

The  ray  which  passes  through  the  center  of  the  lens  (Fig.  44  c) 
and  the  focal  point,  traverses  what  is  termed  the  principal  axis 
of  the  lens.  The  optical  center  of  the  lens  is  a  point  on  the 
principal  axis  at  or  near  the  actual  center  of  the  lens,  through 
which  rays  pass  without  angular  deviation.  Any  line  (e  d),  other 
than  the  principal  axis,  which  passes  through  the  optical  center 
of  the  lens  is  termed  a  secondary  axis. 


Appendix  A :  The  Microscope  and  Its  Optical  Principles    135 


In  the  case  of  a  concave  lens,  parallel  rays  will  be  caused  to 
diverge  (Fig.  45)  and  the  principal  focus,  F,  of  the  lens  is  deter- 
mined by  the  extension  of  the  divergent  rays  till  they  meet  at  a 
point  which  lies  on  the  same  side  of  the  lens  as  the  source  of 
light.  Such  a  point  has  no  actual  existence,  and  is  known,  con- 
sequently, as  a  virtual  focus.  The  focus  of  a  convex  lens,  on  the 
other  hand,  is  real,  and  may  be  determined  readily  by  allowing 
the  sun's  rays,  which  are  prac- 
tically parallel,  to  pass  through 
it  on  to  a  screen.  By  moving 
the  lens  backward  and  forward, 
the  spot  of  projected  light  varies 
in  size  and  brightness.  When 
smallest  and  brightest  the  spot 
is  at  the  focal  point  of  the  lens. 
Images. — In  Fig.  46  the  ob- 
ject, represented  by  an  arrow, 
lies  beyond  the  principal  focus 
of  a  convex  lens  as  in  a  photo- 
graphic camera,  for  example,  or  the  objective  of  a  compound 
microscope.  Light  rays  pass  out  in  all  directions  from  any 
luminous  point.     Hence,  one  ray  from  any  point  on  the  arrow, 

the  tip,  for  instance, 
will  pass  through  the 
focal  point,  F,  and 
one  will  pass  through 
the  optical  center  of 
fig.  46.  the  lens.    From  what 

was  determined  above,  manifestly  the  ray  through  F  will  emerge 
as  one  of  the  parallel  rays  upon  leaving  the  lens,  and  the  one 
through  the  optical  center  of  the  lens,  since  it  traverses  a 
secondary  axis,  will  not  be  refracted,  hence  the  two  rays  must 
cross.  Their  point  of  intersection  is  the  point  at  which  the 
image  of  the  arrow-tip  will  be  formed.  The  same  fact  may  be 
determined,  likewise,  for  any  other  point  of  the  arrow,  for 
example,  the   opposite   end.       Thus  the  distance   from  the  lens 


Fig.  45. 


136  Animal  Micrology 

at  which  the  image  is  formed  may  readily  be  determined.  In 
focusing  a  photographic  camera,  for  example,  the  image  comes 
sharply  into  view  on  the  ground-glass  plate  at  the  back  of  the 
camera  when  the  plate  is  brought  into  the  plane  in  which  these 
rays  through  the  focus  and  the  optical  center  intersect  beyond 
the  lens.  It  will  be  observed  from  the  figure  that  the  image  is 
reversed.  The  size  of  the  image  diminishes  as  the  object  lies 
farther  beyond  F. 

In  case  the  object  lies  between  the  lens  and  the  principal  focus, 
as  in  Fig.  47,  parallel  rays  from  the  object  would  converge  to 
meet  at  the  conjugate  focus  F',  and  an  eye  at  this  point  would 
see  the  image  projected  and  enlarged  without  being  reversed. 
The  plane  in  which  the  image  is  formed  is  determined  by  finding 
the  points  of  intersection  of  the  secondary  axis  through  points  of 
the  object  with  the  imaginary  elongation  of  the  refracted  rays  as 
shown  in  the  figure.  The  image  is  magnified  because  the  observer 
judges  of  the  size  of  an  object  by  the  visual  angle  which  it  sub- 
tends. The  greater  the  convexity  of  the  lens,  the  shorter  the 
focus,  and  also,  since  the  rays  are  bent  more,  the  greater  the 
magnification. 

The  Simple  Microscope. — The  simple  microscope  (the  ordinary 

so-called  magnifiers,  etc.)  operates  upon  this  principle;  the  image 

of  an  object  is  projected  and  enlarged  but  not  inverted  (Fig.  47). 

The  question  arises  as  to  why  there  is  a  best  distance  to  hold 

the  simple  microscope  from  an  object.     Why  will  not  any  point 

answer  so  long  as  it  is  within 
the  focal  point?  As  a  matter  of 
fact,  the  object  may  be  placed 
at  any  point  within  the  focus, 
and  it  will  be  found  that  the 
nearer  it  is  brought  to  the  lens 
the  less  it  is  magnified.  There 
is  one  most  favorable  point  for 
observation,  however,  which  is  neither  at  the  point  of  highest  nor 
of  lowest  magnification,  but  an  intermediate  point,  where  the  lens 
is  freest  from  chromatic  and  spherical  aberrations. 


Fig.  47. 


Appendix  A:  The  Microscope  and  Its  Optical  Principles    137 

The  Compound  Microscope. — The  general  principle  of  the  com- 
pound microscope  is  represented  in  Fig.  48.  The  object  ab 
lies  beyond  the  principal  focus  of  the  first  lens  or  objective  (really 
a  system  of  lenses),  hence  the  image  AB  is  reversed.  This 
image,  in  turn,  is  viewed  through  a  lens,  the  eyepiece  or  ocular 
situated  nearer  the  eye  of  the  observer.  The  ocular  acts  as  a 
simple  magnifier,   projecting  and  enlarging  the  image  but  not 


Fig.  48. 

reversing  it  again.  As  a  matter  of  fact,  the  ordinary  ocular  of  a 
compound  microscope  cannot  be  taken  from  the  instrument  and 
used  as  a  simple  magnifier  because  it  is  made  of  two  planoconvex 
lenses  which  ire  so  adjusted  that  the  image  from  the  objective  of 
the  compound  microscope  is  not  brought  to  focus  until  it  has 
traversed  the  larger  or  field  lens  of  the  eyepiece  (Fig.  52).  The 
image  is  really  examined,  therefore,  at  a  point  between  the  two 
lenses  of  the  eyepiece.  Such  an  eyepiece  is  termed  a  negative 
eyepiece  or  ocular  and  is  widely  used  today  for  microscopical  work. 
Positive  eyepieces  are  made,  however,  and  they  may  be  used  as 
simple  magnifiers  when  removed  from  the  compound  microscope. 
A  good  objective  is  made  up  of  from  two  to  five  systems  of 
lenses  as  shown  in  Fig.  49.     A  single  system  in  turn  may  be  a 


138 


Animal  Micrology 


doublet  (Fig.  54)  or  a  triplet,  each  made  of  •  different  kinds  and 
shapes  of  glass.  A  good  objective  is  a  very  delicate  piece  of 
apparatus  and  must  be  handled  with  great  care.  Each  component 
is  very  accurately  ground  and  the  systems  distanced  with  extreme 
precision  in  order  to  get  a  clear  image.     If  not  already  familiar 


|-inch  Objective.  I-inch  Objective.       TVinch  Oil  Immersion  Objective. 

Fig.  49.— Lens  Systems  of  Various  Objectives. 
Bausch  and  Lomb  §-inch,  J-inch,  and  ^-inch  oil-immersion  objectives  respectively. 

with  the  parts  of  the  compound  microscope,  the  student  should 
study  Figs.  51  and  52  with  a  microscope  before  him. 

DEFECTS  IN  THE  IMAGE 

Spherical  Aberration. — A  simple  convex  lens,  unless  corrected, 
will  not  give  a  sharply  defined  image  because  it  does  not  refract 
to  the  same  degree  all  rays  passing  through  it.  Those  which 
traverse  its  edges  are  brought  to  a  focus  nearer  the  lens  (Fig.  50). 
This  results  not  only  in  an  indis- 
tinct image  but  in  a  distortion  of 
shape  as  well.  Straight  lines,  for 
example,  appear  curved  and  when 
the  parts  of  the  object  are  in  focus 
in  the  center  of  the  field,  those 
nearer  the  margin  are  hazy  and 
indistinct.  This  defect  is  greatest  in  strongly  curved  lenses,  that 
is,  in  high  powers,  since  magnification  increases  with  increased 
curvature.  Spherical  aberration  is  corrected  by  one  or  more  of 
the  following  processes: 


Appendix  A :  The  Microscope  and  Its  Optical  Principles    139 

1.  Cutting  off  the  marginal  rays. 

2.  Changing  the  shape  of  the  surface  of  the  lens. 

3.  Combining  several  lenses  equivalent  to  a  single  lens. 
Chromatic  Aberration. — As  with  a  prism,  ordinary  light  in  pass- 
ing through  a  lens  is  broken  up  into  its  component  colors.      This 


/V. 


... Oeulav\ 


HaaylTSlta^., m 

'  A 

¥a 

V&gfW-- Draw*  tub  a. 

Nosepiece., ^ 

/    w 

H^- Rack*  Rnien»  % 

11  j          Coarse  Adjustment; 

Objectives,^*       ^^^^^ 
Sta9e. ~^       ""-g 

I     A^^HHP>_   Micrometer  Head  or 
fcs.  jB      M       Fine  Adjust raervti 

MS^~Arm. 

li  pper  I  ri  s  Dia  pf,  rag  m  _•       .  .^^^B^g 

Condenser  Mounting -^RSmI 

LQ^er  Iris  Diaphragm.  \J|| 

ME Fine  Adjustment) 

my                 Ptiur. 

■F- Cl£pa. 

W                     ..fVtsmVMliAr, 

Condenser  Focusing  Screw_^fi88 

Hirror .                                  iSff 

Mirror  Fork..^**     jfe^ 

Mirror  Bar _ M &__^^SB 

■k — Horse  Shoe Base. 

Fig.  51.— A  Compound  Microscope  with  Parts  Named. 

process  is  technically  termed  dispersion.  Since  the  colors  are 
not  all  bent  to  the  same  extent,  the  result  is  that  each  color  has  a 
different  focus;  the  ones  which  are  bent  most  (violet  rays)  come 
to  a  focus  nearest  the  lens  and  those  which  are  least  affected  (red 


140 


Animal  Micrology 


Fig.  53. 


rays)  meet  at  a  point  farther  away  (Fig.  53).  This  failure  of  the 
color  rays  to  meet  in  one  focal  point  is  termed  chromatic  aberra- 
tion, and  if  uncorrected  causes  the  image  of  an  object  viewed 
through  such  a  lens  to  be  bordered 
by  a  colored  halo. 

The  defect  is  corrected  by  prop- 
erly combining  glasses  of  different 
dispersive  powers  but  of  kindred 
refractive  powers.  Flint  glass 
(silicate  of  potassium  and  lead), 
for  example,  has  a  dispersive  power  equal  to  about  twice  that  of 

crown  glass  (silicate  of  potas- 
sium and  lime),  although  their 
°Jthfc0cuIar  refractive  powers  are  nearly  the 
same.  By  combining  a  bicon- 
vex lens  of  crown  glass  with  a 
concave  lens  of  flint  glass  so 
constructed  that  its  dispersive 
power  will  just  equal  that  of  the 
crown  glass  (Fig.  54),  the  error 
may  in  large  measure  be  cor- 
rected. Such  an  arrangement 
does  not  interfere  seriously  with 
the  refractive  powers  of  the  lens 
so  constructed.  Unfortunately 
no  two  kinds  of  glass  have  been 
found  which  have  proportional 
dispersive  powers  for  all  colors, 
so  that  in  the  ordinary  achro- 
matic objective  only 
two  of  the  different 
colors  of  the  spec- 
trum have  been  ac- 
curately corrected 
and  brought  to  one 
focus.      The   colors 


-Slide.1 


Coverglas^ 


..Draw  TubeDiaphragm 
with  Society  Screw 


...Society  Screw 


--Mount 

..BaoKlens 

Middle  lens     (?",,, 
.front  lens     \0bje.ettve 
fTJWorkirtgD'retancc, 


Fig.  52.— Sectional  View  of  Microscope  Tube 
including  Ocular  and  Objective. 


Fig.  54. 


Fig.  55.— The  Zeiss  Stand,  IC. 
Equipped  with  Accessories  for  Micro-Photography. 


142 


Animal  Micrology 


Fig.  53.— The  Spencer  No.  25  Stand. 


Appendix  A:  The  Microscope  and  Its  Optical  Principles     143 


Fig.  57.— The  Bausch  and  Lomb  CA  Stand. 


144 


Animal  Micrology 


left  outstanding  form  the  defect  known  as  a  secondary  spectrum. 
In  the  apochromatic  objectives  (p.  146)  three  rays  are  brought  to 
one  focus,  leaving  only  a  slight  tertiary  spectrum. 

NOMENCLATURE  OR  RATING  OF  OBJECTIVES  AND  OCULARS 

Oculars. — Different  makers,  unfortunately,  use  different  sys- 
tems in  marking  their  lenses  to  indicate  relative  powers  of  mag- 
nification.    In  the  case  of  lettering  the  system  is  wholly  arbitrary ; 

the  only  rule  is  that  the 
nearer  to  A  the  letter  is, 
the  lower  the  magnifica- 
tion. When  the  objective 
bears  a  figure  it  is  usually 
indicative  of  the  magnify- 
ing power  of  the  part 
marked.  Thus  a  y1^  inch 
objective  magnifies  ap- 
proximately 120  diamet- 
ers ;  a  J  inch,  80  diameters ; 
a  -J  inch,  20  diameters;  a 
1  inch,  10  diameters:  a  2 
inch,  5  diameters;  and  so 
on.  This  means  that  an 
objective  which  forms  an 
image  10  times  the  real 
diameter  of  the  object 
itself,  on  a  screen  placed 
10  inches  (the  conven- 
tional distance  of  vision) 
from  its  back  lens,  is  rated 
as  a  1-inch  objective.  If  it 
formed  an  image  only  5 
times  the  real  diameter  of 
the  object  it  would  be  a  2- 


FlG. 


58.— The   Zeiss  IIIB   Stand  with   Diaphragm  - 
Carrier  (B)  and  its  Key  (C). 


Zeiss  is  represented  in  America  by  The  Scientific 
Shop,  324  Dearborn  street,  Chicago,  111. 

inch  objective,  if  30  times,  a  J-inch  objective,  and  so  on.  Such 
magnification  is  termed  the  initial  magnifying  power  of  the 
objective. 


Appendix  A :    The  Microscope  and  Its  Optical  Principles      145 

The  •  objectives  of  French  and  German  instruments  are  rated 
in  millimeters  and  the  conventional  distance  of  vision  taken  as 
250  millimeters.  An  objective  of  3  millimeters  focus,  therefore, 
yields  an  initial  magnification  of  83.3  diameters  (^  X  250  =  83.3) . 
Compensating  oculars  (see  below)  bear  numbers  which  indicate 
the  number  of  times  the  eyepiece,  when  used  at  a  given  tube- 
length,  increases  the  initial  magnification.  Ocular  12,  for  exam- 
ple, with  a  3-millimeter  objective  would  yield  a  magnification  of 
83.3X12  =  1,000  diameters,  with  a  standard  length  of  tube. 
Unfortunately  this  simple  system  does  not  apply  to  most  ordinary 
oculars  which  are  more  or  less  arbitrarily  lettered  or  numbered. 

SOME  COMMON  MICROSCOPICAL  TERMS  AND  APPLIANCES 

(Alphabetically  Arranged) 

Achromatic  Objective.  —  An  objective  corrected  for  chromatic  aberration. 
The  correction  is  not  absolute. 

Achromatism.  —  Freedom  from  chromatic  aberration. 

Angular  Aperture. — The  angle  (measured  in  degrees)  formed  at  the 
point  of  focus  (F,  Fig.  59)  by  the  outermost  rays  (a  F,  b  F)  which  trav- 
erse the  objective  to  form  an  image.  This  angle  is  an  important  con- 
sideration because  on  it  depends  in  large  measure  the 
defining  or  resolving  power  of  the  objective.  It  is  evident 
that  the  larger  the  angle  is,  the  greater  the  number  of  rays 
of  light  that  will  be  admitted  from  an  object.  Thus  the 
object  will  be  better  defined  to  the  eye.  In  low  powers 
the  angle  may  be  very  wide,  in  high  powers  it  must 
necessarily  be  small.  Two  objectives,  even  though  they 
may  possess  different  powers  of  magnification,  will  have  the  same  bril- 
liancy if  they  are  of  the  same  angular  aperture;  on  the  other  hand,  if 
they  have  the  same  magnifying  power  but  differ  in  angular  aperture, 
the  brilliancy  is  reduced  in  the  one  of  smaller  angle.  In  immersion 
lenses  the  liquid  used  between  the  lens  and  the  object,  by  reducing 
refraction  has  the  effect  of  increasing  the  angle  of  aperture.  See 
immersion  objective,  also  numerical  aperture. 

Apertometer. — An  instrument  for  measuring  both  the  angular  and  the 
numerical  aperture  of  objectives.  It  is  fitted  to  the  stage  of  the  micro- 
scope. 

Aplanatism. — Freedom  from  spherical  aberration.  The  result  is  a  flat 
field  as  viewed  through  the  microscope.  Aplanatic  lenses  are  usually 
also  achromatic. 


146  Animal  Micrology 

Apochromatic  Objective.  —  An  improved  form  of  objective  which  is  more 
exactly  achromatic  than  the  ordinary  objective  because  it  is  corrected  for 
rays  of  three  colors  instead  of  two,  and  this  correction  is  equally  good  in 
all  parts  of  the  field.  In  the  ordinary  achromatic  objective  after  correc- 
tion there  is  a  residue  of  color  which  is  known  as  the  secondary  spectrum. 
In  the  apochromatic  lenses  correction  is  made  for  a  third  color,  and 
usually  only  a  slight  tertiary  spectrum  is  left  uncorrected.  Spherical 
aberration  is  also  more  fully  corrected.  Furthermore,  in  these  objectives 
the  foci  of  the  optical  and  the  chemical  rays  are  identical,  hence  the 
lenses  are  well  adapted  to  photography  In  the  glasses  of  the  apochro- 
matics,  silicon  is  replaced  by  boron  in  the  flint  series,  and  by  phosphorus 
in  the  crown  series.  Fluorite  was  used  in  conjunction  with  the  glasses 
in  the  earlier  forms  of  apochromatic  lenses,  with  the  result  that  the 
lenses  frequently  deteriorated  in  warm,  moist  climates.  Several  makers 
are  now  able  to  construct  apochromatic  objectives  without  the  use  of 
fluorite.    Both  dry  and  immersion  apochromatics  are  made. 

Binocular  Microscope. — A  microscope  adapted  to  vision  with  both  eyes 
at  once.  By  means  of  a  prism  part  of  the  light  from  the  object  is 
diverted  into  a  second  tube  which  like  the  main  tube  is  provided  with  an 
eyepiece.  Binocular  eyepieces  for  attachment  to  an  ordinary  microscope 
are  now  made.  Binocular  microscopes  yield  a  stereoscopic  view  so  that 
objects  which  have  any  amount  of  depth  stand  out  in  relief  exhibiting 
their  natural  contour.  The  instruments  can  be  used  successfully  only 
with  objectives  of  comparatively  low  power. 

Brownian  Movement  or  Pedesis. — An  oscillating  or  dancing  motion  ob- 
servable in  small  particles  in  a  liquid  when  seen  under  the  microscope. 

Camera  Lucida. — An  apparatus  containing  a  glass  prism  or  thin  glass 
plate  so  arranged  that  when  placed  over  the  eyepiece  of  the  microscope 
the  observer  may  see  the  image  of  the  object  under  the  microscope  pro- 
jected on  to  his  drawing-paper  on  the  table.  The  point  of  the  pencil  is 
also  visible,  consequently  the  outline  of  the  object  may  be  readily  traced 
on  the  paper.  In  the  simpler  camera  lucidas  a  thin  neutral  tint  glass 
slip  is  so  arranged  that  it  is  in  alignment  with  the  eye-lens  of  the  ocular, 
except  that  it  sets  at  an  angle  of  45°  to  it.  When  the  microscope  is 
tilted  into  a  horizontal  position  the  observer  sees  the  image  of  the  object 
reflected  from  the  upper  side  of  the  glass  slip,  but,  since  the  latter  is 
somewhat  transparent,  he  also  sees  the  white  paper  spread  below  on  the 
table  (Fig.  60). 

Another  form  of  simple  camera  lucida  is  the  Wollaston.  To  use 
it  the  microscope  must  be  inclined.  The  essential  part  of  the  camera 
consists  of  a  quadrangular  prism.  The  eye  of  the  observer  is  so  placed 
over  the  edge  of  the  prism  as  to  receive  rays  of  light  from  the  object 


Appendix  A:  The  Microscope  and  Its  Optical  Principles    147 

with  one  portion  of  the  pupil,  and  from  the  drawing-paper  with  the 
remainder. 

Some  form  of  the  Abbe  camera  lucida,  however,  is  used  by  most 
workers.  It  consists  of  a  cap  which  is  fitted  immediately  above  the  eye- 
piece and  which  contains  two  right-angle  prisms  cemented  together  to 


Fig.  60.— Simple  Camera,  Lucida.  Fig.  61. — Camera  Lucida,  Abbe. 

form  a  cube  (Fig.  61).  The  lower  one  of  the  prisms  is  silvered  along  its 
cemented  surface  although  a  small  central  opening  is  left  through  which 
the  object  under  the  microscope  may  be  viewed ;  connected  with  the  cap 
is  an  arm  which  bears  a  mirror  and  this  mirror  may  be  so  adjusted  as  to 
reflect  the  image  of  the  drawing-paper  on  the  table  on  to  the  prisms  from 
one  side.  The  prisms  are  so  set  that  the  silvered  surface  of  the  lower 
one  reflects  this  image  upward  to  the  eye  of  the  observer  which  also,  co- 
incidently,  is  viewing  the  magnified  image  of  the  object  through  the  hole 
in  the  silvering.  When  proper  adjustment  of  the  light  received  from 
object  and  paper  respectively  is  made,  a  pencil  point  may  be  distinctly 
seen  when  brought  into  the  field  of  vision  over  the  paper ;  consequently, 
the  outline  of  the  object  may  be  accurately  traced.  The  secret  of  success 
in  working  with  a  camera  lucida  is  to  have  the  illumination  in  the  two 
fields  properly  balanced.  Small  screens  of  tinted  glass  are  provided 
with  the  instrument  for  such  regulation.  Low-power  eyepieces  should 
be  used.  With  the  Abbe  camera  lucida  the  microscope  may  be  used  in 
a  vertical  or  in  an  inclined  position.  If  the  microscope  stand  is  inclined, 
the  drawing-board  upon  which  the  paper  rests  must  have  the  same  in- 
clination, or  the  outline  when  drawn  will  be  distorted.  Likewise,  if  the 
mirror  of  the  camera  is  at  any  other  angle  than  45  degrees,  an  adjust- 
ment of  the  drawing-surface  must  be  made ;  in  short,  the  axial  ray  of  the 
image  and  the  drawing-surface  must  always  be  at  right  angles  to  pre- 
vent distortion.  This  means  that  if  the  mirror  is  depressed  below  45 
degrees  the  drawing-surface  must  be  tilted  toward  the  microscope  twice 
as  much  as  the  mirror  is  depressed.  For  example,  if  the  mirror  is  de- 
pressed to  37  degrees  (8  below  45  degrees),  the  drawing-board  must  be 
tilted  (raised)  16  degrees.  When  the  camera  is  in  proper  position  the 
field  of  the  microscope  should  appear  at  about  the  same  size  as  without 


148 


Animal  Microlo'gy 


the  camera.  If  the  field  is  reduced  or  unevenly  lighted,  the  camera  is 
too  near  or  too  far  from  the  ocular,  or  it  is  tilted,  or  the  prism  is  not 
properly  centered. 

Fig.  62  represents  a  simpler  form  of  camera  lucida  with  Abbe  prism; 
the  mirror  is  fixed  and  close  to  the  prism. 

Compensating   Ocular.  —  A  specially  designed  eyepiece  for  use   with 
apochromatic  lenses.     It  was  found  -advantageous  to  undercorrect  the 

objective  and  then  to  rectify  the  aber- 
ration by  over-correcting  the  ocular. 
The  so-called  searching  ocular  is  a  low- 
power  compensating  ocular  used  for  the 
first  finding  of  objects.  The  object  once 
located  in  the  field,  the  higher  working 
'$  .  *«J  oculars  are  used  in  observation. 

Condenser. — A  lens  or  a  series  of 
lenses  mounted  in  a  substage  attach- 
ment for  the  purpose  of  concentrating 
light  upon  the  object  to  be  examined. 
They  are  made  in  various  grades  of  ex- 
cellence non-achromatic,  achromatic,  and 
apochromatic.  Some  wide-angle  con- 
densers are  used  as  immersion  conden- 
the  immersion  fluid  is  placed  between  the  upper  surface  of  the 
condenser  and  the  lower  surface  of  the  object  slide.  Condensers  are 
especially  valuable  with  high-power  objectives  and  oil-immersion  lenses. 
They  are  constructed  to  receive  parallel  rays  of  light,  hence  the  plane 
mirror  only  should  be  used  with  them  if  the  illumination  is  from  day- 
light.    See  illumination. 

Correction  Collar. — A  device  for  adjusting  the  distance  between  the 
lens  systems  of  objectives  so  that  the  proper  corrections  may  be  made 
for  different  thicknesses  of  cover-glass.  Low-power  objectives  are  not  so 
sensitive  as  those  of  high  power  to  the  influence  of  the  cover-glass. 
Ordinary  objectives,  however,  are  mounted  in  a  rigid  setting  and  cor- 
rected for  a  specific  tube-length  and  a  standard  cover-glass  (about 
0.18  mm.  thick).  With  a  cover-glass  of  different  thickness  correction 
should  be  made  by  altering  the  tube-length  of  the  microscope,  lengthen- 
ing it  for  a  thinner  cover  and  shortening  it  for  a  thicker  one.  With  homo- 
geneous immersion  lenses  the  defect  caused  by  different  thicknesses  of 
cover-glass  disappears  '(see  immersion  objective).  See  also  tube-length. 
Cover-Glass  Correction. — See  correction  collar. 

Definition. — The  power  of  a  lens  to  give  a  clear,  distinct  image  and 
make  visible  minute  details.     See  resolving  power : 


^bbe  Prism. 


sers: 


Appendix  A :  The  Microscope  and  Its  Optical  Principles    149 

Diaphragm. — Opaque  plates  with  openings  of  various  sizes  for  regu- 
lating the  illumination  of  the  object  to  be  examined.  The  iris  diaphragm 
(Fig.  63)  is  the  best  type.     It  consists  of  a  series  of  overlapping  plates 


Fig.  63. 


-Top  View  of  a  Substage  Attachment  with  Condenser  and  Lower  Iris 
Diaphragm  thrown  out  of  Optical  Axis. 


placed  around  a  central  opening  the  size  of  which  may  be  varied  by 
means  of  a  lever.  Revolving  diaphragms  are  commonly  used  on  the 
cheap  grades  of  microscopes.  They  consist  of  round  disks  perforated 
by  openings  of  various  sizes  which  may  be  rotated  between  the  mirror 
and  the  object.  The  nearer  to  the  object  the  diaphragm  is  placed,  the 
better  the  intensity  of  the  illumination  can  be  regulated.  Most  of  the 
better  class  of  microscopes  are  provided  with  two  iris  diaphragms,  one 
beneath  the  condenser  to  be  employed  when  the  latter  is  in  use,  the 
other  flush  with  the  stage  to  be  used  only  when  the  condenser  is  out. 
If  this  second  iris  diaphragm  is  lacking,  its  place  is  taken  by  means  of  a 
cap-diaphragm  which  may  be  fitted  into  the  substage  in  the  place  of  the 
condenser. 

Dissecting  Microscope. — An  instrument  so  constructed  as  to  enable  an 
operator  to  carry  on  minute  dissections  under  magnification.  Ordinarily 
they  are  simple  microscopes  mounted  on  a  stand  of  some  kind.  The 
best  instruments  (Fig.  64)  are  provided  with  well -corrected  lenses,  with 
glass  stage,  mirror,  black  and  white  substage  plate,  and  rests  for  the 
hands.     See  also  Figs.  65  and  66  for  modified  forms. 

Embryograph. — A  form  of  camera  lucida  for  drawing  at  slight  magni- 
fication small  objects,  such  as  embryos.  A  camera  lucida,  attached  to  a 
simple  microscope  is  frequently  used  for  this  purpose. 

Eye-Point. — The  point  above  an  ocular  or  lens  at  which  the  largest 
number  of  rays  from  the  instrument  enter  the  eye.  The  largest  field  of 
the  microscope  is  visible  from  this  point. 


150 


Animal  Micrology 


Flatness  of  Field. — See  aplanatism. 

Homogeneous  Immersion  Objective. — See  immersion  objective. 

Illumination. — Any  means  employed  to  direct  light  upon  the  object 
under  observation.  Light  which  traverses  the  object  is  said  to  be  trans- 
mitted light.  Most  microscopical  work  in  biology  is  done  by  means  of 
transmitted  light,  hence  the  object  must  be  rendered  more  or  less  trans- 
parent if  not  naturally  so.     If  the  object  is  symmetrically  lighted,  the 


Fig.  64.— Dissecting  Microscope. 

lighting  is  designated  as  axial  or  central  illumination.  If  one  side  is 
lighted  more  than  another,  the  term  oblique  illumination  is  employed. 
In  the  case  of  transmitted  light,  the  light  which  traverses  the  object  is 
usually  light  reflected  from  a  mirror  because  it  is  generally  inconvenient 
or  impossible  to  hold  the  instrument  directly  toward  the  source  of  light. 
Light  which  falls  upon  the  object  and  is  reflected  from  it  to  the  eye, 
either  directly  or  through  a  microscope,  is  termed  reflected  light.  Such 
illumination  is  employed  but  little  in  ordinary  histological  work,  but  it 
is  useful  in  the  examination  of  opaque  objects  such  as  metals,  insects, 


Appendix  A :  The  Microscope  and  Its  Optical  Principles     151 


etc.  The  illumination  may  be  increased  by  means  of  a  bull's  eye  con- 
denser or  a  mirror.  In  some  microscopes  the  mirror  can  be  swung  above 
the  stage  for  the  purpose  of  illumining  an  object  which  is  to  be  studied 
by  reflected  light. 

The  best  light  for  microscopical  work  is  light  reflected  from  white 
clouds.  Direct  sunlight  is  never  used.  The  light  should  come  from  in 
front  of  the  observer  or  from  one  side. 
Various  kinds  of  artificial  light  are  used  for 
microscopical  work,  such  as  an  ordinary 
lamp  with  flat  wick,  the  Welsbach,  or  the 


Fig.  66.— High-Power  Dissecting 
Lens,  Bruecke  Type. 

It  may  be  used  on  the  stand  of 
a  dissecting  microscope  or  in  a 
lens  holder. 


Fig.  65.— Stand  for  Dissecting  Lens. 

ordinary  electric  light.  The  Welsbach  is 
perhaps  the  best.  Whatever  the  source,  the 
rays  must  be  steady  and  brilliant.  If  a 
lamp  with  flat  wick  is  used  greater  bril- 
liancy is  secured  when  the  edge  of  the 
flame  is  turned  toward  the  microscope;  the  object  should  be  lighted 
directly  by  the  image  of  the  flame.  To  do  this  with  low  powers,  the 
lamp  may  have  to  be  turned  so  that  the  flame  is  oblique  to  the  microscope. 
In  artificial  light  the  rays  are  divergent,  not  parallel  as  in  the  case 
of  sunlight,  hence  they  will  not  come  to  focus  at  the  same  point  when 
reflected  from  the  mirror  as  the  latter  do.  This  should  be  corrected  by 
using  a  large  bull's  eye  condenser  between  the  source  of  light  and  the 
mirror,  or  by  sliding  the  mirror  along  the  mirror-bar  farther  away  from 
the  stage  so  that  the  concave  mirror  will  have  a  longer  distance  in  which 
to  bring  the  rays  to  focus.  If  a  substage  condenser  is  used  the  same 
results  may  be  obtained  by  depressing  the  condenser  somewhat  below 
the  level  of  the  stage.  Lamps  made  for  the  microscope  often  have  a 
metal  chimney  with  a  bull's  eye  in  one  side. 


152 


Animal  Micrology 


The  objectionable  yellowness  of  most  artificial  light  may  be  elim- 
inated by  interposing  a  piece  of  green  signal  glass  between  the  lamp  and 
the  microscope.  With  most  microscopes,  round  slips  of  blue  glass  which 
fit  into  the  substage  mechanism  are  supplied  for  this  purpose.  Many  work- 
ers still  employ  as  a  screen  an  ammonia  sulphate  of  copper  solution  in  a 
globular  flask.  To  make  the  solution  dissolve  a  small  amount  of  copper 
sulphate  in  water,  and  add  ammonia.  At  first  a  precipitate  appears,  but 
if  an  excess  of  ammonia  is  added  this  is  dissolved  and  a  transparent 
deep-blue  liquid  results.  This  should  be  diluted  with  water  sufficiently 
to  get  a  blue  of  just  the  proper  depth  to  render  the  transmitted  light 
white  as  seen  through  the  microscope.  The  globular  flask  also  acts  as  a 
condenser. 

Immersion  Objective. — A  kind  of  objective  in  which  a  liquid  is  used 
between  the  front  lens  and  the  cover-glass.  Cedar  oil  is  the  most  widely 
used  medium.  In  as  much  as  the  optical  properties  of  cedar  oil  (refrac- 
tion and  dispersion)  are  almost  the  same  as  crown  glass  it  is  often  termed 
a  homogeneous  immersion  fluid.  A  homogeneous  immersion  lens, 
therefore,  would  be  one  intended  for  use  with  such  a  fluid.  The  advan- 
tage of  an  immersion  over  a  dry  lens  lies  in  the  fact  that,  other  things 
being  equal,  after  leaving  the  cover-glass  rays  which  would  be  so 
refracted  in  a  rarer  medium  like  air  as  to  miss  the  front  end  of  the  objec- 
tive, reach  this  lens  in  the  case  of  immersions  and  traverse  the  objective. 
With  homogeneous  immersions  the  rays  of  light  are  carried  without 
deflection  through  cover-glass  and  fluid  and  into  the  glass  of  the  front 

lens.  Water  has  a  greater  density  than  air 
and  less  than  glass,  hence,  with  a  water 
immersion  more  rays  of  light  reach  the 
front  lens  than  with  a  dry  lens,  and  less 
than  with  a  homogeneous  immersion  lens 
(Fig.  67).  The  effect  of  an  immersion  is 
practically  to  widen  the  angle  of  the  lens 
(see  angular  aperture). 

Magnifying  Power. — The  power  of  a  lens 
to  multiply  the  apparent  dimensions  of  an 
object  viewed  through  it.  It  should  be 
expressed  in  diameters  not  in  areas.  While 
magnifying  power  is  very  important  it  is 
only  so  in  connection  with  resolving  power. 
If  high  power  were  the  only  essential,  a 
series  of  single  lenses  might  be  used.  The 
impossibility  of  using  such  a  series  for  high 
Fig.  67.— (From  Bausch,  "Manipu-    magnification  is  due  to  the  fact  that  proper 

lation  of  the  Microscope.   )  ° 


Appendix  A:  The  Microscope  and  Its  Optical  'Principles     153 

correction  of  aberrations  cannot  be  made,  and  consequently,  a  distinct 
image  cannot  be  obtained.  For  determination  of  magnification  see 
micrometer. 

Mechanical  Stage. — A  stage  attachment  (Fig.  68)  for  the  more  accurate 
manipulation  of  an  object  or  a  series  of  objects  which  must  be  moved 
about  under  the  objective.    The  best  mechanical  stages  are  provided 


Fig.  68.— Attachable  Mechanical  Stage. 

with  scales  and  verniers  so  that  an  object  once  recorded  may  be  easily 
found  again.  They  are  often  very  serviceable,  especially  with  high 
powers. 

Micrometer. — A  scale  for  measuring  objects  under  the  microscope. 
The  stage  micrometer  consists  of  a  finely  divided  scale  ( -rV  and  if »  mm.) 
ruled  on  glass  or  metal.  It  is  commonly  mounted  on  a  glass  slide 
of  standard  size.  To  determine  the  actual  size  of  an  object  with  the 
stage  micrometer,  it  is  most  convenient  to  use  a  camera  lucida.  The 
outline  of  the  object  to  be  measured  is  projected  on  to  a  sheet  of  drawing- 
paper  and  marked  off.  The  object  is  then  replaced  under  the  micro- 
scope by  the  micrometer  and  the  micrometer  scale  is  projected  on  to  the 
paper.     Knowing  the  actual  distance  between  the  lines  on  the  microm- 


154  Animal  Micrology 

eter  scale,  the  magnification  as  well  as  the  real  size  of  the  object  is  readily 
calculated. 

The  size  of  the  image  projected  onto  a  piece  of  drawing  paper  at  the 
level  of  the  table,  however,  does  not  represent  the  true  magnifying  power 
of  the  microscope.  The  latter  is  really  considerably  smaller  if  the  micro- 
scope is  in  a  vertical  position  because  the  magnification  of  a  lens  or  a 
system  of  lenses  is  calculated  in  terms  of  the  conventional  distance  of 
vision  (250  mm.,  see  page  144)  while  the  distance  from  the  ocular  to  the 
table  is  considerably  more  than  250  mm.    Since  the  rays  of  light  diverge 


Fig.  69.— Filar  Micrometer. 

after  leaving  the  ocular,  manifestly,  the  projected  image  will  be  larger  at 
the  level  of  the  table  than  at  a  level  just  250  mm.  from  the  point  of  emer- 
gence of  the  rays  from  the  ocular.  To  determine  the  actual  magnifica- 
tion of  the  microscope,  therefore,  one  would  have  to  bring  the  drawing 
surface  to  within  250  mm.  of  this  point  of  emergence,  sketch  the  pro- 
jected scale  of  the  stage  micrometer  on  the  paper,  and  then,  by  means  of 
an  ordinary  metric  rule,  compute  the  number  of  times  the  divisions  of 
the  micrometer  scale  have  been  magnified.  The  standard  distance  of 
250  mm.,  if  the  Abbe  camera  lucida  is  used  (with  camera  mirror  at  45s), 
includes  the  distance  along  the  mirror-bar  from  the  optical  axis  of  the 
ocular  to  the  mirror,  plus  the  distance  from  the  mirror  to  the  drawing 
surface. 

In  practical  work  it  is  not  necessary  to  make  drawings  or  measure- 
ments exactly  at  this  standard  distance;  one  needs  only  to  have  a  scale 
made  out  for  the  distance  from  the  camera  lucida  at  which  the  drawings 
are  actually  to  be  made,  although  it  must  be  carefully  borne  in  mind 
that  any  variation  in  the  elevation  of  the  drawing  surface  will  alter  the 
size  of  the  projected  image.     A  series  of  carefully  prepared  scales  for 


Appendix  A:  The  Microscope  and  Its  Optical  Principles     155 

various  combinations  of  objectives  and  oculars  should  be  made  and  kept 
for  future  use.  On  each  should  be  recorded  the  tube- length  used,  the 
number  of  the  objective  and  of  the  ocular,  the  length  of  the  camera  mir- 
ror-bar, and  the  angle  of  the  mirror,  for  if  any  one  of  these  is  changed 
the  scale  is  no  longer  accurate. 

When  much  measuring  is  to  be  done  an  ocular  micrometer  is  used. 
It  consists  of  a  circular  glass  disk  with  a  scale  ruled  on  it  and  is  inserted 
in  the  ocular  between  the  eye-lens  and  the  field-lens.  By  means  of  a 
stage  micrometer  the  value  of  the  divisions  of  the  ocular  micrometer  is 
determined  for  a  known  tube-length  and  every  combination  of  lenses  it 
is  desired  to  use  in  the  work  of  measurement.  Suppose  that  it  takes  four 
divisions  of  the  ocular  micrometer  to  correspond  to  one  of  the  finer  divi- 
sions of  the  stage  micrometer,  then  since  the  divisions  of  the  latter  are 
equal  to  tou  mm.,  each  space  in  the  ocular  micrometer  must  be  equal  to 
4- ^  mm.,  that  is  0.0025  mm.  A  filar  or  screw  micrometer  is  a  more  conven- 
ient form  of  ocular  micrometer  which  is  provided  with  delicate  movable 
spider  lines  that  can  be  adjusted  to  the  space  to  be  measured  by  means  of 
a  fine  screw  with  very  accurately  cut  threads  (Fig.  69).  At  the  end  of 
the  screw  is  a  graduated  disk  which  gives  the  value  of  the  distance 
between  the  spider  lines.  The  pitch  of  the  screw  is  either  -fr  inch  or 
0.5  mm. 

Micron. — The  one-thousandth  part  of  a  millimeter;  expressed  briefly 
by  the  Greek  letter  jx.    It  is  the  unit  of  measurement  in  microscopy. 

Mirror. — The  compound  microscope  is  usually  provided  with  both 
concave  and  plane  mirrors,  which  may  be  rotated  or  swung  in  any  direc- 
tion. The  plane  mirror  is  used  with  the  condenser,  the  concave,  when- 
ever it  is  of  advantage  to  have  light  concentrated  upon  the  object  with 
the  condenser  out.  The  mirror  should  be  capable  of  being  moved  up  or 
down  the  mirror-bar  so  that  it  can  be  accurately  focused  upon  the  object. 
See  also  illumination. 

Muscae  Volitantes. — Small  filaments  or  specks  which  float  across  the 
field  of  vision.  They  are  really  small  opacities  in  the  vitreous  humor  of 
the  eye. 

Numerical  Aperture. — A  system  which  expresses  the  efficiency  of  an 
objective  by  indicating  the  relative  proportion  of  light  rays  which  trav- 
erse it  to  form  an  image.  With  the  introduction  of  immersion  objectives, 
it  became  evident  that  angular  aperture  alone  is  not  sufficient  to  indicate 
the  real  capacity  of  an  objective.  For  instance,  an  immersion  and  a  dry 
lens  may  be  of  precisely  the  same  angular  aperture  and  yet  the  immer- 
sion lens  is  more  efficient  because  it  sends  more  rays  of  light  through  the 
objective  (see  immersion  lens).  It  was  found  necessary  to  take  cogniz- 
ance of  the  medium  which  intervenes  between  the  cover-glass  and  the 
front  lens  of  the  objective. 


156  Animal  Micrology 

Professor  Abbe,  in  1873,  proposed  the  name  numerical  aperture  and 
introduced  the  formula  N.  A.=n  sin  u  in  which  n  signifies  the  refractive 
index  of  the  medium  between  cover-glass  and  objective,  and  u  equals 
half  the  angle  of  aperture.  That  is,  by  multiplying  the  refractive  index 
of  the  medium  by  the  sine  of  half  the  angle  of  aperture,  the  numerical 
aperture  is  obtained.  For  example,  suppose  that  one  had  an  oil -immer- 
sion lens  of  90  degrees  angular  aperture,  then  half  the  angle  of  aper- 
ture is  45  degrees,  and  by  turning  to  a  table  of  natural  sines,  the  sine  of 
45  degrees  is  found  to  be  0.707.  The  refractive  index  of  cedar  oil  is  1.52. 
Then  N.  A. =1.52x0.707 =1.075.  Suppose  that  the  lens  were  a  dry  instead 
of  an  immersion  lens;  then  since  the  refractive  index  of  air  is  1,  the  for- 
mula would  read  N.  A.=lx0.707=0.707.  Thus  the  two  products  1.075 
and  0.707  respectively,  represent  the  relative  capacities  of  an  oil  immer- 
sion and  a  dry  objective  of  90  degrees  angular  aperture. 

Parfocal. — A  term  ordinarily  applied  to  eyepieces  of  different  powers 
that  may  be  exchanged  in  the  microscope  without  very  materially  affect- 
ing the  focus  of  the  instrument.  The  term  is  also  applied  to  objectives 
attached  to  a  revolving  nosepiece  if  each  is  approximately  in  focus  when 
turned  into  place. 

Pedesis. — Same  as  Brownian  movement. 

Penetration. — The  quality  of  an  objective  that  permits  of  "looking 
into"  an  object  having  sensible  thickness.  It  is  greatest  with  low  powers 
and  narrow  angles  and  is  antagonistic  to  resolving  power.  It  is  the  nat- 
ural consequence  of  certain  conditions  in  the  making  of  lenses  and  is 
reckoned  of  secondary  importance,  because  practically  the  same  results 
are  obtained  by  manipulating  the  fine  adjustment. 

Polariscope. — As  used  in  microscopy  the  polariscope  consists  of  two 
parts,  each  composed  of  a  Nicol  prism  of  Iceland  spar;  one,  the  polar- 
izer, fits  into  the  substage,  and  the  other,  the  analyzer,  is  inserted  between 
the  objective  and  the  tube  of  the  microscope  or,  in  some  forms,  just  above 
the  ocular.  The  polariscope  is  used  more  in  chemical  and  in  geological 
than  in  histological  work.  Some  of  the  uses  are  as  follows:  determining 
whether  an  object  is  singly  or  doubly  refractive;  detecting  the  presence 
of  minute  crystals;  determining  the  composition  of  rocks;  examining 
sections  of  bone,  hoof  and  horn,  hairs  and  fibers  of  animals  and  plants, 
starch,  etc.,  for  certain  characteristic  and  striking  effects. 

Resolving  Power. — The  quality  of  an  objective  which  enables  the 
observer  to  make  out  fine  details  of  structure.  It  is  the  most  essential 
property  for  precision  in  observation,  and  determines  largely  the  excellence 
of  an  objective.  Resolving  power  depends  upon  careful  correction  of 
aberrations,  general  accuracy  in  the  mechanical  construction  of  the 
microscope,  and  upon  the  aperture  of  the  objective  (see  angular  aperture, 


Appendix  A :  The  Microscope  and  Its  Optical  Principles     157 

numerical  aperture).  Resolving  power  is  tested  by  the  resolution  of  fine 
parallel  lines  ruled  on  glass  or  the  striae  on  the  surface  of  diatoms.  The 
test  is  to  determine  how  many  lines  to  the  inch  or  centimeter  may  be 
distinguished,  and  whether  the  objective  simply  glimpses  the  markings 
or  whether  it  resolves  them  clearly.  The  wider  the  angle  of  aperture,  the 
better  the  resolving  power,  provided  the  width  is  not  so  great  as  to  inter- 
fere with  the  correction  of  the  lenses.  The  increased  resolution  of  immer- 
sion lenses  is  due  to  the  fact  that  the  immersion  fluid  practically  widens 
the  angle  of  aperture  (see  immersion  objective). 

Tube-Length. — The  distance  between  the  places  of  insertion  of  ocular 
and  objective  into  the  tube  of  the  microscope.  There  are  two  standard 
tube-lengths;  the  short  standard  is  160  mm.  (6-fV  inches),  the  long 
standard,  216  mm.  (8-rV  inches).  Many  makers,  however,  do  not  adhere 
to  the  standards.  The  optical  efficiency  of  the  instrument  is  the  same 
in  either  case.  The  short  length  is  more  advantageous  in  that  it  is  more 
compact.  The  lenses  must  be  corrected  for  the  length  of  tube  with 
which  they  are  to  be  used.  The  short  standard  is  in  use  in  most 
American  laboratories. 

Overcorrection  and  Undercorrection. — In  correcting  for  chromatic  aberra- 
tion, if  the  concave  lens  is  stronger  than  is  necessary  to  neutralize  the 
aberration  of  the  convex  lens,  the  blue  rays  are  brought  to  focus  beyond 
the  true  principal  focus  of  the  objective,  and  the  latter  is  said  to  be  over- 
corrected;  if  the  concave  lens  is  not  strong  enough,  the  result  is  what  is 
known  as  undercorrection.  In  case  of  overcorrection,  the  object  takes  on 
an  orange  tint  if,  after  focusing,  the  distance  between  object  and 
objective  is  slightly  increased;  or  it  becomes  of  bluish  color  if  the  distance 
is  decreased.  In  case  of  undercorrection  just  the  reverse  is  true.  In  some 
instances  the  objective  is  purposely  undercorrected,  and  the  eyepiece 
(e.  g.,  compensating  ocular)  is  equally  overcorrected. 

Working-Distance. — The  distance  between  the  front  lens  of  the  objective 
and  the  object  when  the  latter  is  in  focus.  With  high  powers  it  is  very 
small,  so  that  with  some  oil-immersion  objectives  if  a  thick  cover  is  used 
it  is  impossible  to  focus  upon  the  object.  For  this  reason  thin  cover- 
glasses  (No.  1)  should  be  used  on  preparations  which  are  to  be  used  with 
high-power  immersion  lenses. 

MANIPULATION  OF  THE  COMPOUND  MICROSCOPE 

1.  Always  handle  the  instrument  cautiously;  it  is  a  delicate 
mechanism.     Lift  it  by  the  base,  not  by  the  tube  or  the  arm. 

2.  The  work-table  should  be  of  such  a  height  that  the  observer 
can  sit  at  it  comfortably  without  compressing  the  chest  or  tiring 


158  Animal  Micrology 

the  neck.  Sit  as  upright  as  possible.  If  the  instrument  is 
inclined  it  should  set  farther  in  on  the  table  than  if  it  is  in  the 
upright  position. 

3.  With  a  piece  of  old  linen,  a  chamois  skin,  or  a  bit  of  lens 
paper,  carefully  clean  the  eyepiece  to  be  used  and  put  it  in  place. 
Always  use  the  low-power  eyepiece  first. 

4.  Likewise  clean  and  attach  the  objective  (low-power  first) 
after  elevating  the  tube  far  enough  above  the  stage  for  this  pur- 
pose. Guard  particularly  against  screwing  the  objective  in 
crooked,  as  this  will  injure  the  threads.  It  is  best  to  swing  the 
objective  between  the  first  and  second  fingers  of  one  hand  and 
bring  the  screw  squarely  into  contact  with  the  screw  of  the  tube 
(or  nosepiece) ;  with  the  thumb  and  forefinger  of  the  other 
hand  it  is  then  screwed  into  place. 

5.  Bring  the  draw-tube  to  the  standard  length  (see  tube-length) 
for  which  the  lenses  are  corrected.  If  a  nosepiece  is  used,  allow- 
ance must  be  made  for  its  height. 

6.  Place  the  slide  which  bears  the  object  on  the  stage  with 
the  object  over  the  central  opening  of  the  latter,  and  clamp  it  in 
place  by  means  of  the  spring  clips.  While  looking  at  the  object 
from  one  side,  turn  the  mirror  until  a  flood  of  light  shines  up 
through  the  center  of  the  stage. 

7.  Lower  the  tube  until  the  objective  nearly  touches  the  cover- 
glass,  then  look  through  the  eyepiece  and  slowly  raise  the  tube 
by  means  of  the  coarse  adjustment  until  the  specimen  to  be 
examined  is  plainly  visible.  Focus  accurately  by  means  of  the 
fine  adjustment.  If  a  high-power  objective  is  being  used,  since 
it  must  come  very  near  the  cover,  the  operator  should  lower  his 
head  to  the  level  of  the  stage,  and  look  toward  the  light  between 
objective  and  cover-glass  in  order  to  prevent  actual  contact.  This 
is  of  great  importance,  for  otherwise  the  objective  or  the  object 
is  liable  to  injury.  Kemember  that  in  focussing  up  the  lowest 
part  of  the  object  comes  into  view  first,  the  highest  part  last. 

8.  The  higher  the  power,  the  more  difficult  it  is  to  find  an 
object  or  a  particular  part  of  it.  For  this  reason  the  finding  is 
usually  done  by  means  of  a  low-power  objective,  or  a  low-power 


Appendix  A :  The  Microscope  and  Its  Optical  Principles     159 

ocular,  or  both,  and  after  accurately  centering  the  object  in  the 
field,  the  high  power  is  attached.  In  case  a  revolving  nosepiece 
is  used,  great  care  should  be  used  in  turning  in  the  high  power 
not  to  strike  the  slide  with  the  objective.  This  is  very  likely  to 
happen  if  the  objectives  are  not  parfocal. 

9.  After  the  object  is  in  focus  give  any  further  attention  to 
the  illumination  that  is  necessary  (see  illumination  and  mirror). 
If  intensified  illumination  is  desired,  use  the  concave  mirror,  or 
use  the  substage  condenser  and  the  plane-mirror.  For  ordinary 
purposes  the  field  should  be  evenly  illuminated,  although  oblique 
light  is  frequently  useful.  Manipulate  the  diaphragm  until  the 
structure  to  be  studied  shows  with  the  greatest  distinctness.  Too 
much  light  "drowns"  the  object,  and  is  hard  on  the  eyes.  (To 
determine  the  proper  distance  at  which  the  concave  mirror  should 
stand  below  the  stage,  let  direct  sunlight  shine  upon  the  mirror, 
and  then  adjust  the  latter  so  that  the  apex  of  the  cone  of  light 
comes  just  at  the  top  of  the  stage  where  the  object  will  rest.) 

10.  In  using  oil-immersion  objectives,  a  small  drop  of  cedar 
oil  (specially  prepared  by  the  maker  of  the  lens)  is  applied  to 
the  front  lens  by  means  of  a  small  rod  or  brush.  It  is  very 
important  to  keep  the  oil  free  from  dust,  and  to  see  that  it  does 
not  contain  air  bubbles  when  applied  to  the  lens.  Carefully  lower 
the  tube  until  the  oil  on  the  objective  comes  in  contact  with  the 
cover-glass.  The  operator  should  lower  his  head  to  the  level  of 
the  stage  to  observe  this  properly.  Focus  up  as  with  a  dry 
objective.  With  a  piece  of  lens  paper  or  a  soft  cloth,  clean  the 
immersion  lens  immediately  after  you  have  finished  using  it. 
Likewise  remove  the  oil  from  the  cover-glass. 

11.  The  range  of  the  fine  adjustment  is  limited.  Keep  it  as 
near  the  middle  point  as  possible.  If  the  tube  does  not  respond 
to  the  movement  of  the  screw  you  have  probably  gone  beyond  the 
range  of  the  fine  adjustment. 

12.  In  working  with  the  microscope  keep  both  eyes  open.  The 
eye  which  is  not  in  use  soon  becomes  accustomed  to  ignoring  objects 
in  the  field  of  vision.  To  avoid  fatigue  it  is  well  to  use  first  one 
eye  and  then  the  other  for  observation.     The  eye  should  be  placed 


160  Animal  Micrology 

at  the  eyepoint  (see  above)  of  the  lens.  This  is  some  distance 
from  the  eye-lens  in  low-power  eyepieces,  close  to  it  in  high- 
power  eyepieces. 

13.  Put  the  microscope  in  its  case  when  you  have  finished 
using  it,  or  at  least  cover  it  with  a  cloth  or  cone  of  paper.  For 
further  details  regarding  the  use  or  care  of  the  microscope  consult 
one  of  the  following  books:  The  Microscope,  by  Gage;  Manipu- 
lation of  the  Microscope,  by  Bausch;  The  Microscope  and  its 
Revelations  (1,200  pages),  by  Carpenter  and  Dallinger. 

14.  Do  not  apply  alcohol  to  any  part  of  the  instrument.  The 
lenses  may  be  cleaned  ordinarily  by  breathing  upon  them  and 
wiping  them  with  a  rotary  motion  on  lens  paper  or  a  piece  of  soft 
old  linen.  In  case  a  solvent  must  be  used  for  balsam  or  oil,  ben- 
zene is  the  one  commonly  recommended.  It  must  be  quickly 
wiped  away  so  that  it  will  not  affect  the  setting  of  the  lens.  Bits 
of  dust  may  be  flecked  off  the  surface  of  a  lens  by  means  of  a 
camel's  hair  brush. 

The  beginner  in  microscopy  should  acquaint  himself  with 
various  common  objects  that  are  liable  to  get  into  his  preparations 
in  the  form  of  dust,  etc.,  so  that  he  may  not  mistake  them  for  es- 
sential parts  of  his  specimen.  Such  objects  are  hairs,  fibers  of 
silk,  wool,  linen,  cotton,  and  the  like,  and  particularly  air-bubbles. 
Air-bubbles  are  usually  circular  with  black  borders  and  bright 
centers;  they  may  show  tinges  of  color.  Examine  a  drop  of 
saliva  for  examples. 


APPENDIX  B 

SOME   STANDARD  REAGENTS  AND  THEIR  USES 

I.  FIXING  AND  HARDENING  AGENTS 

1.  Acetic  Acid. — Acetic  acid  is  more  commonly  used  in  mixtures 
or  in  diluted  form  than  pure.  It  is  valuable  because  it  tends  to 
produce  good  optical  differentiation  and  facilitates  penetration. 
When  employed  alone  it  causes  some  tissues  to  swell  and  disinte- 
grate. Inasmuch  as  most  fixing  agents  give  the  best  results 
when  they  have  an  acid  reaction,  from  1  to  5  per  cent,  of  acetic 
acid  is  generally  added  to  acidify  them  in  case  they  are  not  nat- 
turally  acid.  Acetic  acid  is  also  of  great  value  in  mixtures  be- 
cause it  counteracts  the  shrinking  action  of  certain  reagents. 
Ordinary  acetic  acid  is  of  about  36  per  cent,  strength;  glacial 
acetic,  of  about  99.5  per  cent,  strength. 

A  strength  of  from  0.2  to  1  per  cent,  is  recommended  by 
Flemming  for  work  on  cell  nuclei.  Strong  glacial  acetic  acid  is 
sometimes  used  for  highly  contractile  animals,  such  as  Coelenter- 
ata,  Mollusca,  and  Vermes.  The  animal  is  rapidly  flooded  with 
the  acid  and  remains  immersed  until  it  is  thoroughly  penetrated 
(6  to  10  minutes).  It  is  then  washed  in  repeated  changes  of  50 
or  70  per  cent,  alcohol  and  left  to  harden  in  70  to  83  per  cent, 
alcohol.  The  pure  acid,  if  allowed  to  act  for  more  than  a  few 
minutes,  swells  and  softens  the  tissues.  Acetic  acid  should  not 
be  used  when  connective  tissue  or  delicate  calcareous  structures 
are  to  be  preserved. 

2.  Acetic  Alcohol. — Carnoy  recommends  each  of  the  following 
formulae : 

a)  Glacial  acetic  acid        1  part 

Absolute  alcohol 3  parts 

b)  Glacial  acetic  acid 1  part 

Absolute  alcohol 6  parts 

Chloroform 3  parts 

The  chloroform  is  said  to  hasten  the  action  of  the  mixture. 
Either  of  these  reagents  penetrates  well  and  acts  rapidly.   Almost 

161 


162  Animal  Micrology 

any  stain  will  follow  them.  Even  such  difficult  objects  as  the 
eggs  of  Ascaris  may  be  fixed  by  the  second  mixture.  The  reagent 
should  be  washed  out  in  absolute  or  at  least  in  strong  alcohol. 

A  mixture  of  absolute  alcohol,  glacial  acetic  acid  and  chloro- 
form, equal  parts,  saturated  with  corrosive  sublimate  (formula  of 
Carnoy  and  Lebrun)  becomes  even  more  valuable  for  the  fixation 
of  difficult  objects.  According  to  Lee,  isolated  ova  of  Ascaris 
are  fixed  in  30  seconds,  entire  oviducts  in  10  minutes,  in  this 
liquid. 

3.  Alcohol. — Alcohol  is  used  especially  for  gland  cells  and  for 
preserving  the  brain  and  spinal  cord  for  Nissl's  method  of  staining 
nerve  cells.  See  chap,  iii,  alcohol  fixation;  also  chap,  i,  reagents 
i  and  2. 

Alcohol  and  Chloroform. — See  2b. 

Bichloride  of  Mercury. — See  corrosive  sublimate. 

4.  Bichromate  of  Potassium. — Bichromate  of  potash  is  one  of 
the  oldest  and  best-known  fixing  reagents.  At  present  it  is  more 
commonly  used  in  mixtures  than  alone.  It  is  widely  used  in 
hardening  nervous  tissue.  Its  fixation  of  nuclei  is  unsatisfactory 
unless  it  is  properly  corrected  through  the  addition  of  acetic  acid. 
It  acts  very  slowly,  about  three  weeks  being  necessary  to  harden 
properly  a  sheep's  eye,  and  from  three  to  six  months  for  a  good- 
sized  brain.  A  weak  solution  (2  per  cent.)  should  be  used  at 
first,  to  be  replaced  gradually  by  stronger  solutions  up  to  5  per 
cent.  When  hardening  is  completed  the  object  should  be  thor- 
oughly washed  in  running  water  and  then  put  into  alcohol ;  begin 
with  low  percentages  of  alcohol  and  gradually  increase  the  strength 
up  to  70  or  80  per  cent.  Change  the  alcohol  as  often  as  it 
becomes  yellow.  After  the  object  has  been  placed  in  alcohol, 
keep  it  in  the  dark  in  order  to  prevent  a  precipitate  forming  on 
the  surface.  Either  carmine  or  hematoxylin  may  be  used  as  a 
stain  after  bichromate  of  potash.  In  case  carmine  is  used,  the 
staining  is  best  done  before  the  object  is  placed  in  alcohol. 
Tissues  which  do  not  stain  well  should  be  placed  for  3  hours  in 
acid  alcohol  and  then  washed  in  alcohol  before  staining. 


Appendix  B:  Some  Standard  Reagents  and  Their  Uses     163 

5.  Bichromate  of  Potassium  and  Acetic  Acid  ( Telly  esnicky's 
fluid).— 

Bichromate  of  potassium     ...*,..      3  grams 

Glacial  acetic  acid 5  c.c. 

Water 100  c.c. 

It  is  best  not  to  add  the  acetic  acid  until  just  before  using. 

This  is   a  good  general  reagent.     It  is  valuable  for  embryos. 

Objects  should  remain  in  some  20  volumes  of  the  fluid  from  24 

to  48  hours,  according  to  size.     It  is  well  to  change  the  fluid 

once,  after  a  few  hours.     After  fixation,  tissues  should  be  washed 

thoroughly  in  running  water  (6  to  12  hours)  and  passed  through 

alcohols  of  increasing  strength  beginning  with  15  per  cent. 

6.  Bichromate  of  Potassium  and  Corrosive  Sublimate  (Zenker's 
fluid).— 

Corrosive  sublimate 5  grams 

Potassium  bichromate 2  grams 

Sodium  sulphate 1  gram 

Glacial  acetic  acid 5  c.c. 

Water 100  c.c. 

It  is  best  to  add  the  acetic  acid  immediately  before  using. 
Zenker's  is  a  valuable  reagent  for  both  histological  and  embryo- 
logical  material  (embryos  up  to  25  mm.).  Several  hours  are 
required  for  fixation:  2  to  4  hours  for  a  2  day  chick ;  8  to  10  hours 
for  objects  or  embryos  of  6  to  8  mm. ;  24  hours  for  embryos  of  12 
to  14  mm.,  etc.  For  washing,  running  water  is  employed  for 
from  12  to  24  hours.  The  object  is  then  transferred  to  gradually 
increasing  strengths  of  alcohol  up  to  70  per  cent.,  leaving  it 
according  to  size  from  1  to  3  hours  in  each  alcohol.  To  remove 
the  excess  of  corrosive  sublimate,  see  13,  caution  1.  Almost  any 
stain  follows  this  reagent  well.  Both  nuclear  and  cytoplasmic 
structures  are  properly  fixed. 

7.  Bichromate  of  Potassium  and  Cupric  Sulphate  (Erlicki's 
fluid). — See  chap,  i,  reagent  8  and  chap,  iii,  3. 

8.  Bichromate  of  Potassium  and  Sodium  Sulphate  (Muller's 
fluid).— 

Bichromate  of  potassium 20  to  25  grams 

Sodium  sulphate 10  grams 

Water 1,000  c.c. 

Muller's  fluid  is  an  old  and  widely  used  reagent.  It  is  espe- 
cially valuable  for    the    nervous  system.     It    acts    very    slowly. 


164  Animal  Micrology 

Specimens  require  immersion  in  a  large  quantity  of  the  fluid  from 
3  to  10  weeks  according  to  size.  The  solution  should  be  changed 
every  two  days  for  the  first  ten  days,  and  later,  about  once  a 
week.  If  a  scum  appears  at  any  time  the  fluid  should  be  changed. 
In  washing,  the  tissues  are  placed  in  running  water  for  a  number 
of  hours  and  are  then  treated  with  gradually  increasing  strengths 
of  alcohol  in  the  usual  manner.  For  some  purposes,  however,  the 
tissue  is  transferred  directly  from  the  fluid  to  70  per  cent,  alco- 
hol. In  any  event,  the  material  should  always  be  kept  in  the 
dark  to  prevent  precipitation. 
Carnoy's  Acetic  Alcohol,  see  2. 

9.  Chloride  and  Acetate  of  Copper  (Liquid  of  Ripart  and  Petit) . — 

Camphor  water 75.0   grams 

Crystallized  acetic  acid 1.0    gram 

Distilled  water 75.0   c.c. 

Acetate  of  copper 0.30  gram 

Chloride  of  copper 0.39  gram 

This  is  a  good  reagent  for  cytological  work  where  objects  are 

to  be  studied  in  as  fresh  a  condition  as  possible.     Methyl  green 

(56)    should   be    used  for    staining.     Only   aqueous  media    are 

employed  with  such  material. 

10.  Chromic  Acid. — Aqueous  solutions  of  from  0.2  to  1  per 
cent,  are  used.  The  acid  is  best  kept  in  the  form  of  a  1  per  cent, 
stock  solution.  Tissues  are  left  in  at  least  fifty  times  their  vol- 
ume of  the  acid  for  from  24  hours  for  small  pieces  to  one  or  more 
weeks  for  larger  ones.  The  objects  are  then  washed  in  running 
water  for  several  hours,  after  which  they  are  treated  with  gradu- 
ally increasing  strengths  of  alcohol.  Do  the  washing  and  dehy- 
drating in  the  dark.  If  sections  of  chromic  acid  material  do  not 
stain  readily,  they  should  be  treated  for  three  hours  with  acid 
alcohol,  washed  out  with  ordinary  alcohol,  and  then  stained. 
Hematoxylin  or  some  of  the  anilins  are  the  best  stains  for  chromic 
material.  Chromic  acid  hardens  much  more  rapidly  than 
bichromate  of  potash.     It  makes  tissues  extremely  brittle. 

11.  Chromo-Aceto-Osmic  Acid  (Flemming's  solution). — 

Chromic  acid,  1  per  cent,  aqueous  solution  .  15  parts 
Osmic  acid,  2  per  cent,  aqueous  solution  .  .  4  parts 
Glacial  acetic  acid 1  part 


Appendix  B:  Some  Standard  Reagents  and  Their  Uses      165 

This  is  the  so-called  "strong "  solution  of  Flemming.  The  mix- 
ture should  not  be  made  until  immediately  before  using,  because 
it  deteriorates  if  allowed  to  stand  for  any  considerable  length  of 
time.  The  fluid  is  valuable  for  cytological  work,  especially  for  the 
study  of  karyokinetic  figures.  Only  small  pieces  of  tissue  should 
be  used,  as  the  reagent  penetrates  poorly.  They  should  remain  in 
the  fluid  for  from  24  to  48  hours  and  then  be  washed  in  running 
water  for  from  6  to  24  hours.  From  water  they  are  transferred 
to  gradually  increasing  strengths  of  alcohol.  Particles  of  fat  are 
blackened  by  the  mixture.  Sections  stain  well  with  safranin  or 
hematoxylin.     Read  the  remarks  on  osmic  acid,  20. 

12.  Chromo-Platinic  Mixture  (Merkel's  fluid). — 

Chromic  acid,  1  per  cent,  aqueous  solution    .     .    25  c.c. 

Platinic  chloride  (PtCl4),  1  per  cent 25  c.c. 

Distilled  water 150  c.c. 

This  is  an  excellent  reagent  for  delicate  objects  such  as  the 

retina.     Objects  are  left  in  it  for  from  a  few  hours  to  several  days. 

Washing  is  done  with  alcohol  of  50  per  cent,  strength   followed 

by  70  per  cent,  alcohol.     Tissues  so  prepared  stain  well. 

13.  Corrosive  Sublimate  (Mercuric  Chloride,  Bichloride  of 
Mercury). — 

Corrosive  sublimate  is  ordinarily  used  as  a  saturated  solution 
in  distilled  water  (about  a  7  per  cent,  sol.)  or  in  normal  saline. 
The  latter  keeps  better  and  contains  a  greater  percentage  of  the 
sublimate.  Corrosive  sublimate  is  an  excellent  and  rapid  fixing 
fluid  for  many  objects  (glands,  epithelia,  etc.).  Objects  should 
remain  in  the  fluid  only  long  enough  to  become  thoroughly  fixed ; 
this  has  been  accomplished  when  they  have  become  opaque 
throughout.  Only  a  few  minutes  or  even  seconds  are  required  to 
fix  very  delicate  objects,  but  denser  tissues  may  require  from  4  to 
24  hours.  The  value  of  the  fluid  is  usually  enhanced  by  the 
addition  of  5  per  cent,  of  glacial  acetic  acid.  Small  pieces  of 
tissue  (not  over  0.6  cm.  in  diameter)  should  be  used  where 
practicable.  Washing  may  be  done  in  running  water  (several 
hours)  or  in  50  to  70  per  cent,  alcohol. 

Cautions. —  (1)  With  corrosive  sublimate  or  mixtures  contain- 
ing it,  the    mercuric  salt  is  often  not  wholly  removed  in  washing. 


166  Animal  Micrology 

If  the  tissues  are  to  remain  several  days  or  weeks  in  alcohol,  the 
alcohol  will  gradually  extract  it.  If  they  are  to  be  used  within  a 
few  days,  however,  it  is  necessary  to  remove  the  excess  of  subli- 
mate by  adding  tincture  of  iodine  to  the  70  per  cent,  alcohol. 
Sufficient  of  the  tincture  is  added  to  give  the  alcohol  a  port- wine 
color ;  as  often  as  the  color  disappears  the  iodine  must  be  renewed. 
After  from  12  to  48  hours  of  this  treatment,  the  iodine  color 
persists  and  the  object  should  then  be  transferred  to  fresh  70  or 
80  per  cent,  which  must  be  renewed  until  it  no  longer  extracts 
iodine  from  the  specimen. 

(2)  In  handling  corrosive  sublimate,  a  glass  or  horn  spoon 
should  be  used  instead  of  a  metal  instrument,  because  it  corrodes 
metal. 

(3)  Use  distilled  water,  not  tap  water,  in  making  an  aqueous 
solution. 

14.  Corrosive  Sublimate  and  Acetic  Acid. — 

Corrosive  sublimate,  saturated  aqueous  solution 

100  parts 

Glacial  acetic  acid 5  to  10  parts 

This  is  an  excellent  reagent  for  embryonic   tissues   and  for 

organs  which  do  not  contain  a  very  great  amount  of  connective 

tissue.     See  remarks  under  13. 

15.  Corrosive  Sublimate,  Nitric  Acid  Mixture  (Gilson's  mercuro- 
nitric  mixture).     See  chap,  i,  reagent  7  and  chap,  iii,  2. 

Erlicki's  Fluid,  see  7. 

16.  Ether  Alcohol. — Equal  parts  of  sulphuric  ether  and  abso- 
lute alcohol. 

Flemming's  Solution,  see  11.' 

17.  Formalin. — See  chapter  i,  reagent  6  and  chap,  iii,  4. 
It  should  be  borne  in  mind  that  formalin  is  a  reducing  agent  and 
will  rapidly  decompose  such  reagents  as  osmic  acid  or  chromic 
acid  if  mixed  with  them. 

18.  Formalin,  Alcohol,  and  Acetic  Acid  (Lavdowsky's  mix- 
ture ) . — 

Formalin,  commercial 10  parts 

Alcohol,  95  per  cent 50  parts 

Glacial  acetic  acid 2  parts 

Distilled  water 40  parts 


Appendix  B:  Some  Standard  Reagents  and  Their  Uses     167 

This  mixture  is  recommended  in  some  cases  for  the  treatment 
of  embryos,  especially  when  the  nervous  system  is  to  be  studied. 
It  penetrates  well  and  preserves  faithfully;  the  alcohol  counter- 
acts the  swelling  effects  of  the  acetic  acid  and  the  formalin. 
Material  may  remain  in  it  without  injury  for  several  days.  The 
fluid  should  sooner  or  later  be  replaced  by  70  per  cent,  alcohol. 
No  preliminary  washing  is  necessary. 

19.  Formol  Sublimate  (Worcester's  fluid). — a)  Make  a  satu- 
rated solution  of  corrosive  sublimate  in  10  per  cent,  formalin. 
This  reagent  is  recommended  by  Raymond  Pearl  (Journal  of 
Applied  Microscopy,  Vol.  VI,  p.  2451)  as  "extremely  satisfactory" 
for  killing  and  fixing  protozoa.  Washing  may  be  done  in  water 
or  4  per  cent,  formalin.  The  material  may  be  preserved  in  4  per 
cent,  formalin  or  carried  up  the  grades  of  alcohol  to  70  per  cent, 
alcohol. 

b)  If  to  9  parts  of  this  formol-sublimate  mixture,  1  part  of 
glacial  acetic  acid  is  added,  Worcester's  formol-sublimate-acetic 
mixture  is  obtained.  Pearl  recommends  this  highly  for  teleost 
eggs  and  for  embryological  material  in  general.  It  will  not  pro- 
duce coagulations  and  cloudiness  in  the  gelatinous  envelopes  of 
amphibian  eggs,  if  thoroughly  washed  out  after  fixing.  Preser- 
vation is  the  same  as  for  a).  Johnson  (Journal  of  Applied  Micro- 
scopy, Vol.  VI,  p.  2652)  also  recommends  this  reagent  very 
highly  for  general  work  except  in  the  case  of  nervous  tissue. 

Personally,  I  have  found  it  advisable  not  to  prepare  either  of 
the  above  mixtures  until  needed  because  the  formalin,  which  is  a 
reducing  agent,  causes  much  of  the  mercuric  salt  to  pass  over 
into  the  insoluble  mercurous  salt. 

Gilson's  Mercuro-Nitric  Mixture,  see  15. 

Hermann's  Fluid,  see  26. 

Kleinberg's  Picro-Sulphuric,  see  25. 

Lavdowsky's  Mixture,  see  18. 

MerkePs  Fluid,  see  12. 

Miiller's  Fluid,  8. 

20.  Osmic  Acid  (really  the  tetroxide  of  osmium  Os04). — 
Osmic  acid  kills  quickly  and  fixes  well.     It  is  exceedingly  vola- 


168  Animal  Micrology 

tile.  The  chief  objections  to  it,  aside  from  its  extremely  poison- 
ous nature,  are  its  poor  powers  of  penetration,  and  the  fact  that 
it  becomes  reduced  in  the  presence  of  the  least  amount  of  dust 
containing  organic  particles.  The  substance  must  be  handled 
with  the  greatest  care,  as  even  the  vapors  are  dangerous.  It  is 
usually  put  up  in  small  quantities  (0.1  to  1  gram)  in  hermetically 
sealed  glass  tubes.  In  making  up  solutions,  the  wrappings  are 
removed  from  such  a  tube  and  the  tube  is  dropped  into  a  reagent 
bottle  where  it  may  then  be  broken  by  means  of  a  glass-  rod. 
Aside  from  its  use  in  mixtures  (see  11  and  26  ),  the  vapor  or  a 
0.05  to  a  1  per  cent,  aqueous  solution  are  commonly  used.  A 
stock  solution  of  1  per  cent,  is  usually  kept  on  hand.  It  must  be 
kept  free  from  dust.  As  the  most  practical  way  of  preventing 
reduction,  Lee  recommends  that  the  osmic  acid  for  ordinary  work 
be  kept  as  a  solution  in  chromic  acid  (a  2  per  cent.  sol.  of  osmic 
acid  in  a  1  per  cent,  aqueous  sol.  of  chromic  acid).  This  solution 
may  be  employed  in  making  up  Flemming's  solution  or  for  the 
purpose  of  fixation  by  means  of  osmium  vapor.  For  vapor  fixa- 
tion, however,  many  workers  prefer  the  vapor  from  the  solid 
crystals. 

To  fix  by  means  of  the  vapor,  the  tissue  is  pinned  to  the  lower 
end  of  a  cork  which  fits  tightly  into  the  bottle  containing  the  osmic 
acid,  or  it  is  suspended  by  a  thread.  Objects  which  will  adhere 
to  a  slide  are  fixed  by  simply  inverting  the  slide  over  the  mouth 
of  the  bottle.  The  time  required  for  such  fixation  varies  from 
thirty  seconds  or  a  few  minutes  for  isolated  cells,  to  several  hours 
for  thicker  objects,  such  as  the  retina.  For  fixing  in  the  solution, 
24  hours  are  required  ordinarily.  Objects  are  then  washed  in 
running  water  for  the  same  length  of  time.  Only  small  or  thin 
pieces  can  be  fixed  by  means  of  either  the  solution  or  the  vapor. 
The  stains  which  follow  osmic  acid  best  are  hematoxylin,  methyl 
green  (for  study  in  aqueous  media),  alum-carmine,  picro-carmine, 
and  safranin. 

21.  Picric  Acid. — A  cold  saturated  aqueous  solution  (about 
1.2  per  cent.)  of  picric  acid  is  commonly  used.  Small  objects 
are  fixed  in  from  a  few  minutes  (infusoria)  to  6  hours;  objects 


Appendix  B:  Some  Standard  Reagents  and  Their  Uses     169 

up  to  1  cm.  in  size,  in  from  24  to  36  hours.  They  may  be  left  a 
much  longer  time,  however,  without  injury.  Large  objects  may 
require  weeks  for  proper  fixation.  After  fixing,  tissues  should  be 
washed  in  70  per  cent,  alcohol  until  the  alcohol  is  no  longer 
colored  by  the  picric  acid.  The  tissue  should  not  pass,  during 
subsequent  treatment  (with  a  few  exceptions  in  case  of  staining), 
into  an  aqueous  medium  or  into  an  alcohol  of  less  than  70  per  cent, 
strength,  because  such  media  seem  to  undo  the  work  of  fixation. 

22.  Picric  Alcohol. — Gage  recommends  a  0.2  per  cent,  solu- 
tion of  picric  acid  in  50  per  cent,  alcohol  as  an  excellent  fixer  and 
hardener  for  almost  any  tissue  or  organ.  Time  required,  1  to  3 
days.  Entire  objects  which  have  been  fixed  in  picric  acid  or 
in  picric  alcohol  stain  readily  in  borax-carmine  or  paracarmine. 

23.  Picro-Acetic. — Saturate  a  1  per  cent,  aqueous  solution  of 
acetic  acid  with  picric  acid.  This  liquid  is  widely  used  as  a 
general  reagent,  and  is  to  be  preferred  for  most  purposes  to 
picric  acid  alone.  For  washing,  etc.,  see  remarks  under  21.  The 
author  has  found  that  his  preparations  are  improved  if  about  25 
parts  of  formalin  are  added  to  each  100  parts  of  the  picro-acetic. 

24.  Picro-Sublimate. — 
RabVs— 

Picric  acid,  saturated  aqueous  solution  ...  1  vol. 
Corrosive  sublimate,  saturated  aqueous  solution  1  vol. 
Distilled  water 2  vols. 

This  mixture  has  been  especially  recommended  for  embryos. 
They  are  left  in  the  fluid  for  12  hours,  then  washed  in  weak 
alcohol  and  transferred  to  gradually  increasing  strengths  of 
alcohol. 

O.  vom  Bath's — 

Picric  acid,  cold  saturated  solution 1  vol. 

Corrosive  sublimate,  hot  saturated  solution  .  .  1  vol. 
Glacial  acetic  acid 0.5  to  1  vol. 

After  fixing  for  several  hours,  transfer  the  material  directly 
into  alcohol. 

25.  Picro-Sulphuric  (Kleinenberg's). — 

Picric  acid,  saturated  aqueous  solution       .     .     98  vols. 

Sulphuric  acid 2  vols. 

Water 200  vols. 


170  Animal  Micrology 

This  is  an  excellent  reagent  for  embryos,  either  for  entire 
mounts  or  for  sectioning.  Chick  embryos  of  24  to  48  hours 
should  remain  in  the  liquid  for  from  2  to  4  hours;  older  embryos, 
for  from  3  to  6  hours.  For  washing,  70  per  cent,  alcohol  is  used. 
It  should  be  changed  (frequently  at  first)  until  the  color  ceases 
to  come  out  of  the  embryos.  Preserve  in  about  80  per  cent, 
alcohol. 

Lillie  recommends  the  addition  of  glacial  acetic  acid  sufficient 
to  make  a  5  per  cent,  solution  of  acetic  acid.  The  reagent,  as 
thus  modified,  certainly  gives  beautiful  results.  For  staining, 
use  Conklin's  picro-hematoxylin  (48). 

26.  Platino-Aceto-Osmic  Mixture  (Hermann's  fluid). — 
Platinum  chloride,  1  per  cent,  aqueous  solution    60  c.c. 
Osmic  acid,  2  per  cent,  aqueous  solution      .     .      8  c.c. 
Glacial  acetic  acid 4  c.c. 

Hermann's  fluid  is  one  of  the  most  valuable  cytological  re- 
agents. Only  small  pieces  of  tissue  should  be  used.  The  washing 
and  subsequent  treatment  are  the  same  as  for  Flemming's  solution 
(11).  For  subsequent  treatment  with  pyrogallol,  see  64.  Read, 
also,  remarks  on  osmic  acid  (20). 

RabPs  Picro-Sublimate,   see  24. 

Rath's  (0.  vom)  Picro-Sublimate,  see  24. 

Ripart  and  Petit,  Liquid  of,   see  9. 

Tellyesnicky's  Fluid,  see  5. 

Worcester's  Fluid,  see  19. 

Zenker's  Fluid,  see  6. 

II.     STAINS 
Read  the  general  statement  about  stains  in  chap  ii. 

27.  Alum  Cochineal. — 

Potassic  alum .      6  grams 

Powdered  cochineal 6  grams 

Distilled  water 90  c.c. 

Boil  for  half  an  hour;  after  the  fluid  has  settled,  decant  the 
supernatant  liquid,  add  more  water  to  it,  and  boil  it  down  until 
only  90  c.c.  of  the  decoction  remains.  Filter  when  cool,  and  add 
a  bit  of  thymol  or  a  little  salicylic  acid  to  prevent  the  growth  of 


Appendix  B:  Some  Standard  Reagents  and  Their  Uses     171 

mold.  Alum  cochineal  is  one  of  the  best  stains  for  entire  objects. 
It  is  easy  to  work  with,  and  does  not  overstain.  The  time  required 
for  staining  is  from  24  to  36  hours  ordinarily.  After  staining, 
the  object  should  be  washed  in  water  for  15  or  20  minutes  to 
extract  the  alum,  which  would  otherwise  crystallize  when  the 
object  is  placed  in  alcohol.  Too  long  an  immersion  in  water  may 
extract  the  stain  to  too  great  an  extent.  From  water  the  object 
should  be  passed  upward  through  the  grades  of  alcohol,  remaining 
about  an  hour  in  each.  The  writer  has  found  alum  cochineal 
especially  valuable  for  flatworms  (tapeworms,  flukes,  etc.)  and 
embryos.  If  it  is  desired  to  use  a  counterstain  with  it,  Lyon's 
blue,  picric  acid,  orange  G,  or  light  green  will  answer. 

28.  Alum  Carmine. 

Powdered  carmine 1  gram 

Ammonia  alum  (2.5  per  cent,  aqueous  solution)  100  c.c. 

Boil  for  20  minutes,  and  filter  when  cool.  The  uses  and 
manipulation  are  the  same  as  for  27.  These  stains  affect  calcareous 
structures  injuriously. 

29.  Anilin  Stains. — Bead  the  general  remarks  about  anilin 
stains  in  chap.  ii.  The  formulae  for  some  of  the  most  important 
are  given  separately  in  this  list  in  their  proper  alphabetical 
position. 

The  dyes  are  dissolved  in  water,  in  alcohol  of  any  desired 
strength,  or  in  anilin  water,  according  as  they  are  soluble  in  these 
media,  or  as  they  meet  the  needs  of  the  operator.  Some  workers 
even  use  some  of  them  as  counterstains  dissolved  in  the  clearing 
fluid.  For  the  study  of  nuclei,  after  Hermann's  or  Flemming's 
fluid  has  been  used  for  fixing,  the  writer  has  found  a  weakly 
alcoholic  anilin  water  solution  to  be  the  most  satisfactory.  As 
cytoplasmic  contrast  stains,  alcoholic  solutions  (in  70  to  95  per 
cent,  alcohol)  have  given  the  best  results.  Anilin  water  is  made 
by  shaking  up  4  c.c.  of  anilin  oil  in  90  c.c.  of  distilled  water  and 
filtering  the  mixture  through  a  wet  filter.  Enough  alcohol  may 
be  added  to  make  it  a  20  per  cent,  alcohol,  if  a  weakly  alcoholic 
solution  is  desired. 

The  length  of  time  which  sections  should  be  immersed  in  the 


172  Animal  Micrology 

stain  varies  from  a  few  seconds  or  minutes  for  some  of  the  dyes 
(especially  when  used  for  cytoplasm)  to  24  to  36  hours  for  others 
(especially  nuclear).  Sections  usually  overstain,  in  which  case 
they  are  differentiated  by  means  of  alcohol,  either  pure  or  slightly 
acidulated  with  hydrochloric  acid.  The  color  is  thus  extracted 
rapidly;  decolorization  should  be  stopped  immediately  after  the 
color  ceases  to  come  from  the  tissue  in  clouds  (20  seconds  to  3 
minutes).  If  acidulated  alcohol  is  employed,  it  must  be  in  much 
weaker  solution  than  that  used  for  extracting  carmines  or  hema- 
toxylins. One  part  of  hydrochloric  acid  to  1,000  of  water  or 
alcohol  is  about  the  correct  proportion.  When  one  desires  to 
study  the  karyokinetic  figures  of  nuclei,  the  acid-alcohol  differen- 
tiation should  be  employed,  but  if  resting  nuclei  are  to  be  studied, 
only  neutral  alcohol  should  be  used. 

30.  Anilin  Blue  and  Orange  G  (Mallory's  connective  tissue 
stain). — 

a)  Double. — 
Solution  I. 

Phosphomolybdic  acid 1.0  gram 

Distilled  water    . 100.0  c.c. 

Solution  II. 

Anilin  blue  (soluble  in  water) 0.5  gram 

Orange  G 2.0  grams 

Oxalic  acid 2.0  grams 

The  tissue  should  be  fixed  in  mercuric  chloride  or  in  Zenker's 
fluid.  Sections  are  first  placed  in  Solution  I  for  one  or  two  min- 
utes and  then  washed  well  in  water.  They  are  then  transferred  to 
Solution  II  for  from  2  to  20  minutes,  washed  in  water,  dehydrated 
rapidly,  and  cleared  in  xylol.  This  is  an  especially  differential 
stain  for  connective  tissue  fibrils  which  are  colored  a  deep  blue. 
Keratin,  blood  corpuscles,  and  usually  muscle  and  some  nuclei, 
are  stained  yellow;  mucin  and  amyloid  substances,  a  light  blue. 

b)  Triple. — Stain  the  sections  from  1  to  3  minutes  in  a  0.1 
to  0.5  per  cent,  aqueous  solution  of  acid  fuchsin  and  wash  them 
in  water  before  treating  with  Solution  I  as  above.  Further 
procedure  is  the  same  as  for  a. 


Appendix  B:  Some  Standard  Reagents  and  Their  Uses     173 

31.  Bismarck  Brown. — Boil  one  gram  of  the  stain  in  100  c.c. 
of  water,  filter,  and  add  30  c.c.  of  strong  alcohol.  Bismarck 
brown  is  a  nuclear  stain  which  does  not  overstain  although  it  acts 
rapidly.  After  staining  wash  in  95  per  cent,  or  absolute  alco- 
hol. This  stain  is  also  used  in  aqueoas  solution  for  intra  vitam 
staining;  the  nucleus  of  the  living  cell  may  thus  be  colored.  It 
has  been  used  as  an  intra  vitam  stain  mostly  in  the  study  of 
infusoria.  The  stain  may  be  fixed  by  means  of  a  0.2  per  cent, 
chromic  acid  solution,  but  this,  of  course,  destroys  the  life  of  the 
cells. 

32.  Borax-Carmine    (Grenacher's). — See   chap,  i,  reagent  12. 

33.  Bordeaux  Red. — See  chap,  i,  reagent  15. 

34.  Carmalum.  (Mayer's). — 

Carminic   acid 1  gram 

Alum 10  grams 

Distilled  water 200  c.c. 

Dissolve  with  heat  and  filter  the  solution  when  cold.  Add  a 
few  crystals  of  thymol  or  a  little  salicylic  acid  to  prevent  the 
formation  of  mold.  Carmalum  is  one  of  the  best  stains  for  stain- 
ing objects  in  bulk  and  will  follow  almost  any  fixing  reagent, 
even  osmic  acid.  If  the  object  has  an  alkaline  reaction  it  does 
not  stain  so  well.     Washing  is  done  in  water. 

35.  Carmine  (Beale's). — 

Powdered  carmine 1  gram 

Ammonia *'....     3  c.c. 

Pure  glycerin 96  c.c. 

Distilled  water 96  c.c. 

Alcohol,  95  per  cent 24  c.c. 

The  ammonia  and  part  of  the  water  are  first  mixed  and  the 
carmine  dissolved  in  the  mixture.  The  remaining  water  is  added 
and  the  solution  is  left  in  an  open  dish  until  the  ammonia  has 
almost  evaporated.  The  alcohol  and  glycerin  are  then  added. 
For  staining,  equal  parts  of  the  stain  and  glycerin  are  used.  The 
staining  is  carried  on  for  24  hours  under  a  bell  jar  in  an  uncovered 
dish.  A  second  open  dish  containing  acetic  acid  is  placed  under 
the  bell  jar.     After  staining,  the  sections  are  washed  in   water, 

0 


174  Animal  Micrology 

then  in  weak  hydrochloric  acid  (1  to  500  of  water)  and  again  in 
water.  Minot  recommends  this  stain  and  method  of  treatment 
especially  for  the  placenta  and  for  the  central  nervous  system  of 
embryos. 

36.  Carmine,  Picric  Acid,  and  Indigo  Carmine  (Calleja's  staining 
fluid).— 

Solution  I. 

Carmine 2  grams 

Lithium  carbonate,  saturated  aqueous  solu- 
tion   100  c.c. 

Solution  II. 

Indigo  carmine       .  0.25  gram 

Picric  acid,  saturated  aqueous  solution   .     100.00  c.c. 

Place  sections  in  solution  I  for  from  5  to  10  minutes,  then 
into  acid  alcohol  until  they  become  pale  red  (20  to  30  seconds) ; 
wash  well  in  water.  Next,  place  the  sections  in  Solution  II  for  5 
to  10  minutes,  then  into  acetic  acid  (0.2  to  0.5  per  cent.)  for  a 
few  seconds  and  wash  well  in  water.  Dehydrate  rapidly  and 
clear  in  xylol.  The  method  is  useful  for  epithelial  cells  and 
connective  tissue. 

37.  Carmine,  Acid  (Schneider's). — Add  carmine  to  boiling 
acetic  acid  of  45  per  cent,  strength  until  no  more  will  dissolve. 
Filter  the  solution  when  cool.  This  is  a  valuable  reagent  for 
the  study  of  the  nuclei  of  fresh  cells.  It  is  very  penetrating  and 
gives  a  brilliant  stain.  The  strong  acetic  acid  ultimately  destroys 
the  cell. 

38.  Ehrlich-Biondi  Triple  Stain  (Heidenhain).— The  ingre- 
dients should  be  obtained  from  Grubler  and  Hollborn,  Baiersche 
Strasse  63,  Leipzig,  or  from  their  agents. 

Acid  fuchsin,  saturated  aqueous  solution    .    .  4  parts 

Orange  G  "  "  "  .     .  7  parts 

Methyl    green    (Methylgriin    OO)    saturated 

aqueous  solution 8  parts 

The  solution  of  orange  should  be  prepared  first,  and  the  solu- 
tions of  fuchsin  and  methyl  green  added  to  it  with  continual 
stirring.     Each  solution  must  be  thoroughly  saturated ;  it  takes 


Appendix  B:  Some  Standard  Reagents  and  Their  Uses     175 

several  days  for  this  to  occur.  The  above  mixture  constitutes  a 
stock  solution  which  should  be  diluted  with  about  50  or  100  times 
its  volume  of  water  before  using.  According  to  Lee  (Microt- 
omisfs  Vade-Mecum),  "if  a  drop  be  placed  on  blotting  paper  it 
should  form  a  spot  bluish  green  in  the  center,  orange  at  the 
periphery.  If  the  orange  zone  is  surrounded  by  a  broader  red 
zone,  the  mixture  contains  too  much  fuchsin."  For  use  with  this 
method,  tissues  should  be  fixed  in  pure  corrosive  sublimate  solu- 
tion. Sections  should  be  thin  (3  to  5  microns)  and  must  remain 
in  the  stain  from  18  to  24  hours.  They  should  then  be  rapidly 
washed  in  95  per  cent,  alcohol,  placed  for  a  short  time  in  absolute 
alcohol  and  cleared  in  xylol.  If  the  sections  remain  in  the  alco- 
hols any  considerable  length  of  time,  the  methyl  green  will  be 
extracted.  The  stain  is  very  uncertain  in  its  action  but  when 
successfully  applied  the  results  are  excellent.  It  is  used  chiefly 
in  cytological  studies,  especially  in  connection  with  gland  cells. 
Grubler  prepares  a  dry  powder  for  this  three-color  mixture,  but 
the  results  are  usually  not  as  satisfactory  as  when  the  mixture  is 
properly  made  fresh.  To  prepare  the  stain  from  the  powder,  a 
0.4  per  cent,  solution  of  the  latter  in  distilled  water  is  made, 
and  to  100  c.c.  of  this  solution  7  c.c.  of  a  0.5  per  cent,  aqueous 
solution  of  acid  fuchsin  are  added. 

39.  Ehrlich's  "Triacid"  Mixture. — For  blood  films  Ehrlich's  so- 
called  triacid  mixture  is  a  serviceable  stain  which  is  widely  used. 

Orange  G,  saturated  aqueous  solution  .     .     .  14.0  c.c. 

Acid  fuchsin,  saturated  aqueous  solution        .  7.0  c.c. 

Distilled  water 15.0  c.c. 

Absolute  alcohol 25.0  c.c. 

Methyl  green,  saturated  aqueous  solution      .  12.5  c.c. 

Glycerin 10.0  c.c. 

Each  solution  must  be  thoroughly  saturated  (several  days).  Add 
the  ingredients  in  the  order  named,  shaking  the  mixture  well 
between  each  addition.  It  is  best  for  the  stain  to  stand  a  week 
or  two  before  it  is  used.  Neutrophil  granules  stain  violet,  oxy- 
phil granules  a  brownish  red.  The  mixture  stains  in  from 
5  to  15  minutes. 

40.  Eosin. — See  chap,  i,  reagent  17.     This  anilin  dye  is  often 
used  after  hematoxylin  as  a  contrast  stain.      It  is  specific  for  cer- 


176  Animal  Micrology 

tain  granules  of  leucocytes  and  for  red  blood  corpuscles,  giving  to 
the  latter  a  very  characteristic  coppery-red  tinge.  Some  workers 
prefer  to  dissolve  it  in  water  or  in  some  cases  in  the  clearer. 

41.  Erythrosin. — An  eosin ;  properties  and  manipulation,  much 
the  same  as  ordinary  eosin  (see  40). 

42.  Fuchsin,  Acid  (Rubin  S,  Acid  Majenta,  Majenta  S). — 

Acid  fuchsin     .         0.5  gram 

Distilled  water 100.0  c.c. 

This  is  an  excellent  anilin  stain  for  cytoplasmic  structures.  It 
is  also  used  in  some  instances  as  a  specific  stain  for  nerve  tissue. 
Acid  fuchsin  should  not  be  confounded  with  basic  fuchsin  which 
is  a  nuclear  stain.  It  too  is  used  in  aqueous  solution.  When 
fuchsin  alone  is  mentioned  by  writers,  without  specifying  whether 
it  is  acid  or  basic,  the  basic  fuchsin  is  ordinarily  meant. 
43.  Fuchsin  (Acid)  and  Picric  Acid  (Van  Gieson's  stain).— 

Acid  fuchsin,  1  per  cent,  aqueous  solution    .     .     10  c.c. 
Picric  acid,  saturated  aqueous  solution    .     .     .    90  c.c. 

This  stain  is  frequently  used  in  conjunction  with  hematoxylin 
in  the  study  of  fibrous  or  of  nerve  tissue.  Small  bits  of  tissue 
should  be  fixed  in  corrosive  sublimate  or  its  mixtures.  Sections 
are  slightly  overstained  with  hematoxylin,  rinsed  in  water,  and 
then  stained  5  minutes  in  the  picro-fuchsin  mixture.  To  avoid 
extracting  too  much  of  the  yellow  color  in  dehydrating  and  clear- 
ing, the  alcohols  and  clearer  should  each  have  a  few  crystals  of 
picric  acid  added  to  them.  The  result  should  be:  nuclei  and 
epithelia  brown ;  white  fibrous  connective  tissue  red ;  elastic  tissue 
and  muscle  yellow. 

44.  Gentian  Violet. — This  is  one  of  the  best  of  the  nuclear 
anilin  stains.  It  is  best  made  up  in  anilin  water  and  weak 
alcohol  (see  29). 

Gentian  violet        1  gram 

Anilin  water 80  c.c. 

Alcohol,  95  per  cent 20  c.c. 

The  stain  works  well  with  thin  sections.  It  is  also  widely 
used  in  the  study  of  bacteria.  For  differentiation,  Gram's 
method  is  used. 


Appendix  B:  Some  Standard  Reagents  and  Their  Uses     177 

Gram's  solution. — 

Iodine 1  gram 

Iodide  of  potassium 2  grams 

Water 300  c.c. 

After  staining,  the  sections  are  placed  in  this-  solution  until 
they  are  black  (2  to  3  minutes)  and  are  then  decolorized  in  abso- 
lute alcohol  until  they  appear  gray.     See  also  66. 

45.  Gold  Chloride. — The  gold  chloride  method  is  used  chiefly 
in  the  study  of  nerve-fiber  terminations,  both  motor  and  sensory, 
although  it  is  sometimes  used  for  the  coloration  of  other  tissue 
elements  (capsules  of  cartilage,  etc.).  The  process  is  really  an 
impregnation;  through  the  agency  of  sunlight  and  of  certain 
reagents  (acetic,  citric,  formic  or  oxalic  acid)  the  gold  is  deposited 
in  the  tissues  in  the  form  of  very  fine  particles.  There  are  numer- 
ous modifications  of  the  method,  one  of  which  is  given  in  chap.  ix. 

46.  Golgi's  Chrome-Silver  Method. — See  chap.  ix. 

47.  Hemalum  (Mayer's). — 

Hematein     .         1  gram 

Alcohol,  95  per  cent.     . 50  c.c. 

Alum        50  grams 

Distilled  water 1000  c.c. 

Dissolve  the  hematein  in  the  alcohol  with  the  aid  of  heat. 
Dissolve  the  alum  in  the  water  and  slowly  add  the  hematein  solu- 
tion; thoroughly  stir  the  resulting  mixture.  Filter  if  there  is 
any  residue  of  solid  material,  and  add  a  few  crystals  of  thymol  to 
prevent  the  formation  of  mold.  The  stain  may  be  used  imme- 
diately after  preparation.  It  is  valuable  for  staining  in  bulk, 
because  it  does  not  overstain,  especially  if  diluted  one-half  with 
distilled  water.  Large  objects  will  require  at  least  24  hours  of 
staining.  After  staining,  tissues  are  washed  in  water  thoroughly 
to  insure  the  removal  of  all  alum.  If  a  purely  nuclear  stain  is 
desired,  2  per  cent,  of  glacial  acetic  acid  may  be  added  to  the 
hemalum  solution. 

Hematoxylin. — For  general  statement  see  chap.  ii. 

48.  Hematoxylin,  Conklin's  Picro-. — 

Delafield's  hematoxylin 1  part 

Water 4  parts 

Add  one  drop  of  Kleinenberg's  picro-sulphuric  (25)  to  each 


178  Animal  Micrology 

cubic-centimeter  of  the  solution.     This  is  a  beautiful  stain  (1  to  3 
hours)  for  embryos  which  are  to  be  mounted  entire. 

49.  Hematoxylin,  Delafield's. — See  chap,  i,  reagent  13. 

50.  Hematoxylin,  Ehrlich's. — 

Hematoxylin 2  grams 

Absolute  alcohol 100  c.c 

Glacial  acetic  acid 10  c.c. 

Glycerin 100  c.c. 

Distilled  water 100  c.c. 

Ammonia  alum 

Mix  the  glycerin  and  the  water  and  thoroughly  saturate  the 
resulting  fluid  with  the  alum.  The  solution  must  be  exposed  to 
light  and  air  at  least  3  weeks  to  ripen.  It  is  not  ready  for  use 
until  it  acquires  a  deep  red  color.  This  solution  is  an  excellent 
nuclear  stain  and  will  keep  for  years. 

51.  Hematoxylin,  Heidenhain's  Iron. — See  chap,  i,  reagent  18, 
and  chap,  vi,  iv.  This  stain  is  used  chiefly  in  the  study  of  cell 
structures  such  as  centrosomes,  chromosomes,  etc.  Tissues  are 
best  fixed  in  some  of  the  sublimate  solutions  or  in  acetic  alcohol, 
although  it  will  follow  liquid  of  Flemming  or  Hermann.  Sec- 
tions should  be  not  over  6  microns  thick.  The  ferric  solution 
must  be  renewed  occasionally  as  it  soon  spoils. 

52.  Hematoxylin,  Weigert's. — This  method  together  with  its 
modifications  is  a  very  important  one  for  the  study  of  the  tracts 
of  medullated  nerve  fibers. 

Solution  I. 

Neutral  acetate  of  copper,  saturated  aqueous 

solution 1  part 

Distilled  water 1  part 

Solution  II. 

Hematoxylin    . 1  gram 

Absolute  alcohol 10  c.c. 

Lithium  carbonate,    cold  saturated   aqueous 

solution 1  c.c. 

Distilled  water 90  c.c. 

Solution  III. 

Ferricyanide  of  potassium 2.5  grams 

Borax 2.0  grams 

Distilled  water 200.0  c.c. 


Appendix  B:  Some  Standard  Reagents  and  Their  Uses     179 

Harden  the  nervous  tissue  in  Muller's  (8)  or  Erlicki's  (7) 
fluid  and  without  previous  washing  continue  the  hardening  in 
alcohol.  The  hardening  complete,  imbed  in  celloidin  and  place 
the  celloidin  block  into  Sol.  I  for  36  to  48  hours.  Place  it  next 
into  70  to  80  per  cent,  alcohol  for  24  hours ;  the  block  may  be 
left  in  such  alcohol  indefinitely.  Cut  sections  in  the  usual  way 
(not  over  20  to  25  microns)  and  stain  them  in  Sol.  II  for  24 
hours  at  room  temperature,  and  then  for  a  few  hours  in  a  warm 
chamber  at  40°  C.  Wash  the  sections  in  water  and  differentiate 
(a  few  minutes)  in  Sol.  Ill  until  the  gray  matter  (ganglion  cells, 
etc. )  of  the  tissue  becomes  yellow.  The  medullary  sheath  remains 
dark.  Rinse  in  water,  dehydrate,  clear  in  carbol-xylol,  and 
mount  in  balsam.  This  method  may  be  used  also  for  demon- 
strating degenerated  fibers;  they  remain  unstained. 

53.  Light  Green  (Lichtgriin  S.  F.).— This  is  a  beautiful  cyto- 
plasmic anilin  stain  which  is  frequently  used  after  safranin  as  a 
counterstain.  Not  more  than  0.5  per  cent,  solution  should  be 
used  as  it  stains  very  rapidly  and  very  deeply.  It  may  be  used 
either  as  an  aqueous  or  as  an  alcoholic  solution.  The  writer  has 
found  a  0.5  per  cent,  solution  in  95  per  cent,  alcohol  very  satis- 
factory.    Sections  should  remain  in  it  only  a  few  seconds. 

54.  Lyons  Blue  (Bleu  de  Lyon). — This  is  one  of  the  best  of 
the  numerous  anilin  blues.  It  is  a  good  contrast  stain  when 
used  after  such  nuclear  stains  as  safranin  and  carmine.  See 
chap,  i,  reagent  16. 

Mallory's  Connective  Tissue  Stain. — See  30. 
Magenta,  Acid. — See  42. 

55.  Methylen  Blue. — This  reagent  is  an  extremely  useful 
one;  it  is  of  great  value  in  the  study  of  the  nervous  system, 
and  it  can  be  made  to  give  results  with  intercellular  cement 
substance,  lymph  spaces,  etc.,  as  satisfactory  and  with  greater 
certainty  than  impregnations  obtained  with  gold  chloride  or 
silver  nitrate.  It  is  also  serviceable  as  an  intra  vitam  stain. 
Furthermore,  methylen  blue  (saturated  solution  in  70  per  cent, 
alcohol)  followed  by  eosin  is  sometimes  used  for  the  double 
staining  of  blood  corpuscles.  Methylen  blue  should  not  be 
confounded  with  methyl  blue. 


180  Animal  Micrology 

Ordinary  commercial  methylen  blue  usually  contains,  in  addi- 
tion to  the  blue  dye,  a  small  quantity  of  a  reddish-violet  dye.  Such 
methylen  blue  is  termed  polychromatic  and  is  especially  service- 
able in  staining  certain  cell  granules.  Only  the  pure  methylen 
blue,  however,  should  be  used  for  nerve  staining  and  other  intra 
vitam  work. 

a)  Intra  Vitam  Stain  for  Small,  Comparatively  Transparent 
Aquatic  Organisms. — Add  sufficient  methylen  blue  to  the  water 
containing  the  organisms  to  tinge  it  a  light  blue.  Different  tis- 
sues will  take  up  the  color  after  different  intervals  of  time,  and  a 
given  tissue  after  having  attained  a  maximum  degree  of  colora- 
tion will  rapidly  lose  its  color  again.  It  is  necessary,  therefore, 
to  watch  the  organisms  closely  for  the  maximum  of  color  in  the 
tissue  desired.  If  the  observer  wishes,  the  stain  may  be  fixed 
for  more  prolonged  study  by  following  the  processes  indicated 
under  b).  The  order  in  which  various  tissues  take  the  stain 
seems  to  vary  in  different  organisms.  Usually  gland  cells  stain 
first,  then  with  more  or  less  deviation,  other  epithelial  cells,  fat 
cells,  blood  and  lymph  cells,  elastic  fibers,  smooth  muscle,  and 
striated  muscle.  Nerve  cells  and  nerve  fibers  do  not  ordinarily 
take  the  stain  when  the  entire  animal  is  immersed. 

b)  Ehrlich's  Method  for  Nerve-Terminations  and  the  Relations 
of  Nerve  Cells  and  Fibers  to  the  Central  Nervous  System. 
—The  stain  should  be  Grtibler's  methylen  blue  (rectificiert 
nach  Ehrlich ) .  A  1  per  cent,  solution  in  normal  saline  is  used 
Warm  the  solution  till  it  steams,  stir  it  thoroughly  and  when 
cool,  filter.  The  tissue  must  be  perfectly  fresh.  Chloroform 
the  animal  and  immediately  inject  the  stain  into  the  main 
artery  of  the  part  to  be  investigated.  If  the  animal  is  small, 
the  entire  body  may  be  injected.  The  vessels  should  be  filled 
full  but  care  must  be  taken  not  to  rupture  them.  The  part 
should  become  decidedly  blue  in  color.  It  is  well  after  10  or 
15  minutes  to  inject  more  stain.  At  the  expiration  of  half  an 
hour  after  the  second  injection  remove  small  pieces  of  tissue 
containing  the  nerve  elements  desired,  and  expose  them  freely 
to  the  air  on  a  slide  wet  with  normal  saline.  Examine  every 
two  minutes   under  the   microscope   (without  cover-glass)   until 


Appendix  B:  Some  Standard  Reagents  and  Their  Uses     181 

the  particular  element  to  be  investigated  (cell,  axone,  termina- 
tion) lias  developed  a  well-marked  blue  color.  It  is  important 
to  catch  the  color  at  the  proper  stage  and  fix  it  because  it 
soon  begins  to  fade. 

Fixing  the  Stain. — When  the  desired  element  has  developed 
a  satisfactory  blue  color,  the  tissue  is  transferred  immediately  to 
a  saturated  aqueous  solution  of  Ammonium  picrate  (Dogiel's 
method)  and  left  for  from  6  to  24  hours.  For  final  mounting  the 
tissue  should  be  teased  out  sufficiently  to  show  the  proper  ele- 
ments and  then  mounted  in  a  few  drops  of  a  mixture  of  pure  gly- 
cerin (free  from  acid)  and  ammonium  picrate  (saturated  aqueous 
solution),  equal  parts.  It  is  well  to  let  the  tissue  stand  in  20  to 
30  volumes  of  this  glycerin-picrate  mixture  for  a  day  or  two  before 
mounting  it.  If  the  preparation  is  to  be  kept  the  cover-glass 
should  be  sealed  (chap,  xiii,  II,  A,  6). 

Sections. — If  it  is  desired  to  make  paraffin  sections  and  mount 
them  in  balsam,  after  treatment  with  the  ammonium  picrate  (10 
to  15  minutes),  the  tissue  must  be  placed  into  20  or  30  volumes 
of  Bethe's  fluid,  which  renders  the  color  insoluble  in  alcohol. 

BetJie^s  fluid. — 

Molybdate  of  ammonia 1  gram 

Chromic  acid,  2  per  cent,  aqueous  solution     .  10  c.c. 
Hydrochloric  acid,  concentrated  C.  P.     ...     1  drop 
Distilled  water 10  c.c. 

The  tissue  is  left  in  this  mixture  for  from  45  to  60  minutes 
(for  small  objects)  and  then  washed  1  to  2  hours  in  distilled 
water.  Dehydrate  directly  in  absolute  alcohol;  follow  this  with 
xylol,  imbed  in  paraffin,  and  section  in  the  ordinary  manner. 
Sections  may  be  counterstained  in  alum-carmine  or  alum-cochineal. 

c)  Immersion  Method. — Material  which  cannot  be  readily 
injected  or  which  has  failed  to  stain  may  be  stained  by  immersion. 
A  0.1  per  cent,  solution  of  the  stain  is  used  (dilute  1  volume  of 
the  solution  used  for  injection  with  9  volumes  of  normal  saline). 
To  small  pieces  (2  to  3  mm.  thick)  of  the  tissue,  add  a  few 
drops  of  th6  stain  at  intervals  of  about  three  minutes.  The  tis- 
sue should  always  be  moist,  but  never  covered  sufficiently  by  the 
solution  to   exclude  air.     Examine  the  preparation  from  time  to 


182  Animal  Micrology 

time  under  the  microscope  and  when  the  nerve  elements  are  well 
stained,  fix  in  ammonium  picrate  and  proceed  as  in  b).  In  case  of 
the  central  nervous  system,  fairly  good  results  may  sometimes  be 
obtained  by  dusting  the  methylen  blue  powder  over  the  freshly 
cut  surface  of  the  part  to  be  studied.  The  development  and 
fixing  of  the  color  is  the  same  as  in  b ) . 

d)  NissPs  Method  of  Staining  Basophil  (Tigroid)   Substance  in 

Nerve  Cells. — 

Methylen  blue 3.75  grams 

Venetian  soap  (white  castile  soap)     .     .         1.75  grams 
Water 1,000.00  c.c 

It  is  best  to  keep  the  stain  for  some  months  before  using. 

Ganglia  should  be  fixed  in  alcohol,  formalin  or  corrosive  subli- 
mate and  sectioned  in  paraffin.  Fix  the  sections  to  the  slide,  dis- 
solve out  the  paraffin  with  xylol,  and  run  the  preparation  down  to 
the  aqueous  stain  in  the  ordinary  way.  In  a  test-tube  heat  a  few 
cubic  centimeters  of  the  stain  until  it  steams,  then  apply  it  while 
still  warm  to  the  sections  on  the  slide,  which  has  been  placed  flat 
on  the  desk.  It  takes  about  6  minutes  for  the  stain  to  act.  Pour 
off  the  surplus  stain  and  rinse  the  slide  in  distilled  water.  Lay 
it  flat  on  the  desk  again  and  flood  the  sections  with  anilin-alcohol 
(95  per  cent,  alcohol,  9  parts;  anilin  oil,  1  part).  Let  the  sec- 
tions decolorize  (20  to  30  seconds)  until  they  are  a  pale  blue; 
then  drain  off  the  anilin-alcohol  and  transfer  the  preparation  to 
absolute  alcohol.  Clear  in  xylol  and  mount  in  balsam.  The 
basophil  granules  should  appear  deep  blue  in  color.  They  are 
arranged  for  the  most  part  concentrically  around  the  nucleus. 

e)  Unna's  Method  of  Staining  Unstriated  Muscle  in  Sections. — 
Stain  in  a  1  per  cent,  aqueous  solution  of  polychromatic  methylen 
blue,  rinse  in  water  and  then  leave  for  10  minutes  in  a  1  per  cent, 
aqueous  solution  of  potassium  ferricyanide.  Transfer  to  acid 
alcohol  until  sufficiently  decolorized,  then  complete  the  dehydra- 
tion and  mount  in  the  usual  way. 

/)  For  Ordinary  Section  Staining  where  a  nuclear  stain  is 
desired,  methylen  blue  answers  very  well.  It  is  usually  used  (2 
to  24  hours)  in  aqueous  solution.  The  treatment  is  the  same  as 
for  safranin. 


Appendix  B:  Some  Standard  Reagents  and  Their  Uses     183 

g)  Impregnation  of  Epithelia,  etc. — Place  the  fresh  tissue, 
preferably  a  thin  membrane,  into  a  4  per  cent,  solution  of  meth- 
ylen  blue  in  normal  saline.  To  demonstrate  the  outline  of  cells, 
leave  the  tissue  in  the  stain  not  longer  than  10  minutes.  To  get 
a  negative  image  of  lymph  spaces,  canals,  etc.,  in  contrast  to  the 
ground  substance  which  becomes  deeply  impregnated,  leave  the 
tissue  in  the  stain  20  to  30  minutes.  For  this  purpose  it  is 
advisable  to  remove  any  membranous  covering  which  invests  the 
organ.  In  either  case,  after  staining,  fix  the  tissue  for  30  to  40 
minutes  in  a  saturated  aqueous  solution  of  ammonium  picrate, 
changing  it  once  or  twice,  and  examine  in  dilute  glycerin.  To 
preserve  the  preparation  permanently,  proceed  as  in  6).  To  do 
away  with  the  macerating  action  of  the  ammonium  picrate,  add  2 
per  cent,  of  a  1  per  cent,  osmic-acid  solution  to  the  fixing  bath. 

56.  Methyl  Green. — This  is  one  of  the  best  of  the  nuclear 
anilin  stains.  It  is  particularly  valuable  because  it  instantly 
stains  the  chromatin  of  nuclei  in  fresh  tissues.  Use  in  strong 
aqueous  solutions,  acidulated  to  about  1  per  cent,  with  acetic  acid. 
It  does  not  give  a  satisfactory  chromatin  stain  if  the  tissue  has 
been  fixed  in  acetic  acid  or  mixtures  containing  it.  It  follows 
pure  corrosive  sublimate  solution  admirably. 

57.  Methyl  Violet. — This  stain  is  commonly  used  in  0.5  to  2 
per  cent,  aqueous  solutions  for  staining  bacteria,  nuclei,  and  amy- 
loid.    It  may  often  be  substituted  for  gentian  violet. 

58.  Neutral  Red. — Neutral  red  is  used  widely  as  an  intra  vitam 
stain.  It  is  a  good  stain  for  cytoplasmic  granules,  and  in  some 
cases  for  mucus  cells.  For  intra  vitam  staining  it  may  be  used  in 
the  same  way  as  methylen  blue  (with  the  omission  of  fixation). 
For  staining  fixed  material,  a  1  per  cent,  or  stronger  aqueous 
solution  is  employed.  Granules  are  stained  orange  red  (bright 
red  in  acid  medium,  yellow  in  alkaline  medium).  Rosin  finds 
that  in  nerve  cells  stained  in  neutral  red  (followed  by  water,  acid- 
free  alcohols,  xylol,  and  balsam)  nucleoli  and  Nissl's  granules 
are  stained  red,  the  rest  of  the  cell  yellow. 

59.  Orange  G. — This  is  an  excellent  cytoplasmic  stain  and  is 
often  used  on  sections  as  a  contrast  to  carmine,  hematoxylin,  and 
safranin.     Grubler's  Orange  G  is  the  most  reliable.     It  should  be 


184  •       Animal  Micrology 

used  in  saturated  aqueous  solution.     The  solution  does  not  keep 
very  well. 

60.  Orcein  (Unna's  method  for  elastic  fibers). — 

Orcein  (Grtibler's) 1  gram 

Hydrochloric  acid 1  c.c. 

Absolute  alcohol 100  c.c. 

Sections  are  stained  in  a  watch-glass  or  porcelain  dish.  The 
dish  is  warmed  over  a  flame  or  in  an  oven  until  the  stain 
becomes  thick  through  the  evaporation  of  the  alcohol.  Rinse  the 
stained  sections  thoroughly  in  alcohol,  clear  in  xylol  and  mount 
in  balsam.     Elastic  fibers  should  appear  dark  brown. 

61.  Paracarmine  (Mayer's). — 

Carminic  acid 1.0  gram 

Aluminium  chloride 0.5  gram 

Calcium  chloride 4.0  grams 

Alcohol,  70  per  cent 100.0  c.c. 

Paracarmine  is  an  excellent  stain  for  large  objects.  It  does 
not  overstain  ordinarily.  The  stained  tissue  is  washed  in  70  per 
cent,  alcohol.  In  case  overstaining  occurs  add  2.5  per  cent  gla- 
cial acetic  acid  or  0.5  per  cent,  aluminium  chloride  to  the  alcohol 
used  for  washing.  Objects  to  be  stained  should  not  have  an 
alkaline  reaction  nor  contain  limy  materials. 

62.  Picric  Acid. — Picric  acid  is  widely  used  as  a  contrast  stain 
with  carmine,  hematoxylin,  etc.  It  is  best  manipulated  as  a  stain 
by  adding  a  little  to  each  of  the  alcohols  used  in  dehydrating, 
after  application  of  the  nuclear  stain.  However,  if  acid  alcohol 
is  to  be  used,  the  picric  acid  should  be  used  only  in  the  grades 
above  the  acid  alcohol.  It  may  be  employed  in  staining  entire 
objects  as  well  as  sections.     See  also  remarks  on  washing  under  21. 

63.  Pier o-Car mine. — This  is  a  very  useful  double  stain.  Tissues 
stained  in  it  should  never  be  washed  in  water  or  a  low  percentage 
of  alcohol  because  these  extract  the  yellow  color  very  rapidly. 
If  the  tissues  are  to  be  mounted  in  glycerin  or  glycerin  jelly, 
transfer  them  to  the  mounting  fluid  without  washing;  if  they  are 
to  be  mounted  in  balsam,  to  prevent  the  extraction  of  the  stain, 
picric  acid  should  be  added  to  each  of  the  alcohols  through  which 
the  material  will  pass.  Picro-carmine  solutions  are  likely  to  dis- 
solve off  sections  which  have  been  affixed  by  means  of  albumen 


Appendix  B:  Some  Standard  Reagents  and  Their  Uses     185 

fixative.  There  are  various  formulae  for  making  the  stain.  One 
of  the  oldest  and  at  the  same  time  one  of  the  best  is  the  formula 
of  Ranvier. 

Ranvier^s  formula. — A  saturated  solution  of  carmine  in  ammo- 
nia is  added  to  a  saturated  aqueous  solution  of  picric  acid  to  the 
point  of  saturation  (that  is,  until  precipitation  begins).  The 
mixture  is  next  evaporated  down  to  one-fifth  of  its  volume  and 
filtered  after  cooling.  The  solution  is  then  evaporated  until  only 
a  powder  remains.  A  1  per  cent,  solution  of  this  powder  in  dis- 
tilled water  is  used  for  staining.  It  is  well  to  allow  the  stain  to 
act  for  from  12  to  24  hours.  If  the  material  is  to  be  mounted  in 
glycerin,  it  should  first  be  treated  (by  irrigation  under  the  cover- 
glass)  with  formic-glycerin  (formic  acid  1  part,  glycerin  100 
parts)  for  several  days  until  proper  differentiation  has  taken  place. 
If  osmic  acid  has  been  used  for  fixing,  nuclei  should  appear  red, 
muscle  tissue  straw  yellow,  elastic  fibres  canary  yellow,  connec- 
tive tissue  pink,  and  keratohyalin  red. 

64.  Pyrogallol. — Tissues  which  have  been  fixed  in  Hermann's 
or  in  Flemming's  fluid  for  24  to  36  hours  may  be  treated  (with- 
out previous  washing)  with  a  weak  solution  of  pyrogallol  or 
with  crude  pyroligneus  acid.  Lee  (Microtomisf  s  Vade-Mecum) 
recommends  the  pyrogallol  as  much  preferable.  Tissues  should 
remain  in  the  fluid  from  1  to  24  hours  depending  upon  size.  The 
result  is  a  black  stain  which  colors  both  nucleus  and  cytoplasm. 
If  desired,  an  additional  chromatin  stain  may  be  employed. 
Safranin  (65)  for  24  hours  is  recommended;  decolorize  slightly 
with  very  dilute  acid  alcohol.  The  stain  is  excellent  for  cy to- 
logical  work  (for  "sphere,"  etc.). 

65.  Safranin. — Safranin  is  one  of  the  most  important  of  the 
basic  anilin  dyes.  Read  carefully  the  remarks  on  anilin  stains 
under  29. 

Safranin 1  gram 

Anilin  water  (see  29) 90  c.c. 

Alcohol,  95  per  cent 10  c.c. 

Filter  before  using.  Grtibler's  "Safranin  O"  is  the  most  reli- 
able dye.     Sections  of  tissues  fixed  in  Hermann's  or  Flemming's 


186  Animal  Micrology 

solution  are  left  in  the  stains  for  from  24  to  48  hours.     Decolorize 
as  directed  under  29. 

66.  Safranin  and  Gentian  Violet. — This  is  a  combination  that 
is  almost  indispensable  in  the  study  of  cell  problems,  especially 
spermatogenesis.  For  formulae  of  stains  see  44  and  65.  Tissues 
are  best  fixed  in  Flemming's  or  Hermann's  solutions.  Stain  thin 
sections  for  36  to  48  hours  in  the  safranin;  differentiate  in  alco- 
hol very  slightly  acidulated  (see  29),  then  stain  for  5  to  10  min- 
utes in  the  gentian  solution  and  transfer  the  sections  to  Gram's 
solution  (see  under  44)  for  1  to  3  hours.  Finally  differentiate  in 
absolute  alcohol.  As  soon  as  purple  clouds  have  ceased  to  come 
from  the  sections  in  absolute  alcohol,  they  should  be  transferred 
to  clove  oil  for  a  few  minutes  and  thence  to  xylol.  The  clove  oil 
seems  to  intensify  the  safranin  in  the  chromatic  granules,  but  too 
prolonged  an  immersion  in  clove  oil  extracts  the  gentian  violet. 

67.  Silver  Nitrate. — The  nitrate-of -silver  method  is  used  largely 
as  an  impregnation  method  for  work  on  nerve  tissue  and  for  dem- 
onstrating intercellular  substances  and  outlining  boundaries  of 
cells  in  the  epithelial  coverings  of  membranes,  etc.  Wash  the 
fresh  tissue  in  distilled  water,  then  place  it  for  2  to  5  minutes  in 
0.5  to  1  per  cent,  aqueous  solution  of  silver  nitrate.  Rinse  in 
distilled  water,  then  expose  the  tissue  to  bright  sunlight  in  water 
or  glycerin  (or  in  70  per  cent,  alcohol,  if  it  be  mounted  in  balsam) 
until  a  brown  coloration  appears.  Temporary  mounts  should  be 
made  in  glycerin.     For  application  to  nerve  see  chap.  ix. 

68.  Sudan  III. — This  is  a  specific  stain  for  fat.  A  saturated 
alcoholic  solution  is  used  (5  to  10  minutes).  Wash  rapidly  in 
alcohol.  Since  alcohol  is  a  solvent  of  fat,  too  long  an  immersion 
will  destroy  the  preparation.  Mount  in  glycerin.  With  this  dye 
large  fat  drops  stain  orange,  small  ones  yellow.  The  tissue  should 
have  been  fixed  previously  in  Mullers  fluid  (8)  or  other  medium 
which  does  not  dissolve  fat. 

Van  Giesen's  Stain. — See  43. 

69.  Wrights  Stain  (for  blood  and  for  the  malarial  parasite). — 
See  chap,  xiv,  memoranda  5  and  6. 


Appendix  B:  Some  Standard  Reagents  and  Their  Uses     187 

III.     NORMAL  OR  INDIFFERENT   FLUIDS 

(For  fresh  tissues) 

70.  Aqueous  Humor. — Obtained  by  puncturing  the  cornea  of  a 
freshly  excised  beef's  eye.  A  small  amount  may  readily  be 
obtained  by  means  of  a  capillary  pipette  from  the  eye  of  a  freshly 
killed  frog. 

71.  Blood  Serum. — Blood  is  allowed  to  clot  and  after  24  hours 
the  serum  is  poured  off.  If  necessary  it  may  be  further  freed 
of  blood  cells  by  means  of  a  centrifuge.  The  serum  will  keep  for 
only  a  day  or  two.  Schultze^s  iodized  serum  made  by  saturating 
blood  serum  with  iodine  is  sometimes  classed  as  an  indifferent 
fluid,  but  it  is  really  a  dissociating  fluid. 

72.  Fluid  of  Ripart  and  Petit. — 

Camphorated  water       ,     .     .    75.0  c.c. 

Acetate  of  copper 0.3  gram 

Chloride  of  copper 0.3  gram 

Distilled  water 75.0  c.c. 

Glacial  acetic  acid 1.0  c.c. 

After  the  solution  becomes  clear  (a  few  hours)  it  should  be 

filtered.     It  is  especially  useful  for  examining  fresh  animal  cells. 

Methyl  green  is  an  excellent  stain  to  follow  this  fixing  fluid. 

73.  Kronecker's  Fluid. — 

Distilled  water 100.00  c.c. 

Sodium  chloride 0.60  gram 

Sodium  carbonate 0.06  gram 

74.  Normal  Saline. — A  0.7  per  cent,  solution  of  sodium  chloride 
in  distilled  water. 

IV.  DISSOCIATING  FLUIDS 

75.  Bichromate  of  Potassium. — A 0.2  percent,  aqueous  solution 
is  commonly  used.  Nerve  cells  of  the  spinal  cord  and  also  vari- 
ous epithelia  dissociate  well  in  it  (2  to  3  days). 

76.  Caustic  Potash. — A  solution  of  35  parts  in  100  parts  of 
water  is  often  used  for  isolating  fibers  of  smooth  muscle  or  heart 
fibers.  It  acts  by  rapidly  destroying  the  connective  tissue  (20  to 
30  minutes).  Examination  of  the  tissue  is  made  by  mounting  it 
in  the  dissociating  fluid.  If  water  is  added  the  tissue  will  be 
destroyed.     Usually    only   temporary   preparations  are   made  in 


188  Animal  Micrology 

this  fluid,  but  tissues  may  be  made  permanent  by  neutralizing  the 
alkali  by  means  of  acetic  acid. 

77.  Gage's  Formaldehyde  Dissociate*. — See  chap,  i,  reagent  10. 
For  method  of  using  see  chap,  x,  A. 

78.  Hertwig's  Macerating  Fluid. — See  chap,  x,  C. 

79.  Landois*  Solution. — 

Neutral    ammonium    chromate,   saturated 

solution        5  grams 

Potassium  phosphate,  saturated  solution    .  5  grams 

Sodium  sulphate,  saturated  solution       .     .  5  grams 

Distilled  water        100  c.c. 

This  solution  is  valuable  for  the  central  nervous  system.  Small 
pieces  of  tissue  are  placed  in  the  fluid  for  1  to  5  days.  For  stain- 
ing after  maceration,  it  is  recommended  that  the  material  be 
placed  for  24  hours  in  ammonia  carmine  diluted  with  one  volume 
of  the  macerating  fluid. 

80.  MacCallum's  Macerating  Fluid. — 

Nitric  acid 1  part 

Glycerin 2  parts 

Water 2  parts 

This  fluid  is  recommended  for  heart  muscle  of  adults  or 
embryos.  Hearts  should  remain  in  it  from  8  hours  to  3  days, 
according  to  size.  The  method  is  valuable  for  showing  the 
arrangement  of  cardiac  muscle  fibers. 

81.  Ranvier's  One-Third  Alcohol. — This  is  one  of  the  common- 
est as  well  as  one  of  the  best  macerating  fluids.  It  is  simply  a 
30  per  cent,  alcohol.  Epithelia  will  macerate  in  it  sufficiently 
in  24  hours.  A  still  weaker  alcohol  (20  to  25  per  cent.)  is  used 
for  isolating  the  nerve  fibers  of  the  retina. 

82.  Schiefferdecker's  Fluid.— 

Methyl  alcohol 5  c.c. 

Glycerin     " 50  c.c. 

Distilled  water 100  c.c. 

This  fluid  is  used  for  the  retina  and  central  nervous  system. 
It  should  be  prepared  fresh  before  using  and  tissues  must  remain 
in  it  for  several  days. 

83.  Sodium  Chloride. — A  10  per  cent,  solution  of  sodium  chlo- 
ride  is  excellent  for  tendon,  etc.     It  dissolves  the   cement  sub- 


Appendix  B:  Some  Standard  Reagents  and  Their  Uses     189 

stance  of  epithelial  cells  and  of  connective  tissue.  As  a  stain, 
a  saturated  aqueous  solution  of  picric  acid  (stain  for  24  to  36 
hours)  followed,  after  thorough  washing  in  water,  by  a  dilute 
alcoholic  solution  of  acid  fuchsin  gives  excellent  results. 

V.  DECALCIFYING  FLUIDS 

84.  Chromic  Acid. — Chromic  acid  diluted  to  1  per  cent,  or  in 
combination  with  other  fluids  is  frequently  used  for  decalcifica- 
tion. Chromic  acid,  1  gram ;  water,  200  c.c. ;  nitric  acid,  2  c.c,  is  a 
mixture  widely  used.  It  decalcifies  well  but  acts  more  slowly 
than  the  10  per  cent,  nitric  acid  mixture.  Bone  should  first  be 
hardened  in  M  tiller's  fluid  (8). 

85.  Nitric  Acid. — A  10  per  cent,  solution  of  nitric  acid  in  70 
per  cent,  alcohol  may  be  used.  If  nitric  acid  is  used  for  young 
or  foetal  bones,  it  is  advisable  to  use  only  1  part  of  the  acid  to  99 
parts  of  the  alcohol.  After  washing  out  in  70  per  cent,  alcohol, 
the  decalcified  bone  may  be  kept  in  95  per  cent,  alcohol. 

86.  Phloroglucin  Method. — This  is  a  rapid  method.  Young 
bones  may  be  decalcified  in  half  an  hour  and  old  and  hard  ones 
in  a  few  hours.  Teeth  require  a  somewhat  longer  time.  Phloro- 
glucin itself  does  not  decalcify,  but  protects  the  tissue  from  the 
action  of  the  strong  nitric  acid.  One  gram  of  phloroglucin  is 
dissolved  in  10  c.c.  of  pure  non-fuming  nitric  acid  with  the  aid 
of  gentle  heat.  Ten  c.c.  of  nitric  acid  in  100  c.c.  of  water  is  added 
to  the  mixture. 

87.  Picric  Acid. — A  solution  kept  fully  saturated  is  useful  for 
delicate  bones.  It  stains  and  decalcifies  the  tissue  at  the  same 
time.     Wash  in  70  per  cent,  alcohol. 

88.  Von  Ebner's  Fluid.— 

Alcohol,  95  per  cent 500.0  c.c. 

Water 100.0  c.c. 

Sodium  chloride 2.5  grams 

Hydrochloric  acid 5.5  c.c. 

This  is  an  excellent  fluid  for  bone  because  in  it  the  ground 
substance  of  the  bone*  does  not  swell  up.  Sections  are  best 
examined  in  a  10  per  cent,  solution  of  sodium  chloride. 


190 


Animal  Micrology 


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i 
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a: 

15 

Mount  in  melted  Canada  balsam    free 
from  xylol.    Granula  appear  red  in  yel- 
lowish field.    It  is  questionable  if  they 
should  be  regarded  as  vital  structures 

It  is  best  to  clear  sections  in  oil  of  ber- 
gamot  or  other  clearer  having  a  low 
index  of  refraction 

The  cells  possess  very  large  nuclei  and 
nucleoli.    The  glands  may  also  be  fixed 
in  Flemming  (11),  sectioned  in  paraffin 
and  stained  in  Ehrlich-Biondi  (38) 

For  making  permanent  preparations  of 
protozoa,  see  Appendix  I),  \e.    See  also 
Appendix  B,  19a 

20  g.  fuchsin  S  (42)  in 
100  c.c.  of  anilin  wa- 
ter (29) ;    pour  on  to 
slide  and  heat  over 
flame  till  stain  begins 
to  steam.  Wash  with 
picric     acid     (con- 
centrated    ale.     sol. 
plus   2  vols,  water) ; 
add  fresh  picric  and 
heat  for  1  min.  over 
flame 

Iron-hematoxylin  (51) 

Safranin,  light  green 
(65,  53) 

Methyl  green  (56) 

For  intra  vitam  stain- 
ing of  protozoa,  etc., 
see  31,  55a,  or  58 

Section  or  Iso- 
lation Method. 
P.  =  Paraffin ; 
C.=Celloidin; 
F .—  F  reez- 
ing;  H.=Free 
Hand 

P.    Very    thin 
(1-2    /*)     sec- 
tions 

P.    Very    thin 
(1  n)  sections 

p. 

Fixing  and  Hard- 
ening,   or    Other 
P  r  elimmary 
Treatment 

Fix  for  24  hours  in 
potassium    bi- 
chromate (5  p.  c. 
aq.  sol.)  and  os- 
mic  acid  (2  p.  c. 
aq.  sol.),  eq'ual 
parts 

Picric  acid  (21) 

Flemming  (11) 

Examine    in    the 
blood  of  the  larva 

Examine  protozoa 
in  water ;  the  oth- 
ers   in    normal 
saline  (74),  or  bet- 
ter,   in    fluid    of 
Ripart  and  Petit 
(70) 

■41 

ill 

1*1 

III 

Various  organs 

Blood  cells  or  intes- 
tinal epithelium  of 
frog 

Young  ovarian  egg  of 
teleosts 

Testis  of  salamander 

Pull   off    the    larva's 
head ;     the   salivary 
glands     (two     small 
transparent    bodies) 
remain  attached 

Protozoa  and  blood 
corpuscles.  Ovarian 
ova  (teased) 

Scrapings  from  roof  of 
frog's  mouth 

1 
i 

5 

1 

Bioblasts  (granula) 
of  Altmann 

Foam  structure 

o 

a 
1 

1 
g 

a  « 

Gland  cells  of  Chi- 
ronomous  larva 

Living  or  fresh  cells 

Appendix  C:  Table  of  Tissues  and  Organs 


193 


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APPENDIX  D 

PREPARATION  OF  MICROSCOPICAL  MATERIAL  FOR  A 
GENERAL  COURSE  IN  ZOOLOGY 

(In  addition  to  the  methods  enumerated  here,  see  also  chap,  x,  II  and 
chap,  xiii.) 

PROTOZOA 

a)  Cultures. — Amebae,  etc.,  may  usually  be  obtained  in  quan- 
tities sufficient  for  class  use  by  the  following  method  recommended 
by  H.  S.  Jennings: 

A  number  of  glass  dishes  measuring  8  or  9  inches  in  diameter 
by  3  inches  deep  are  crowded  full  of  water  plants  (especially 
Ceratophyllum  and  Elodea),  filled  with  water,  and  the  plants 
allowed  to  decay.  Keep  the  dishes  in  warm,  light  places.  In 
two  or  three  weeks  the  layers  of  plants  at  the  surface  of  the  water 
will  be  covered  with  a  brown  slime  which  should  be  examined 
occasionally  under  the  microscope  for  the  desired  forms.  The 
scum  that  appears  on  the  surface  of  the  water  consists  mainly  of 
bacteria  upon  which  amebae  largely  feed.  They  will  be  found 
most  frequently  in  the  slime  that  immediately  surrounds  the  plant 
tissue.  Since  they  frequently  last  only  two  or  three  days  in  a 
culture,  to  insure  material  for  class  work,  a  number  of  cultures 
must  be  made  at  different  dates  and  from  different  localities. 
Other  protozoa  such  as  Arcella,  Difflugia,  Carchesium,  Stenior, 
etc.,  will  also  be  found  in  the  cultures. 

Paramoecium  may  be  kept  from  dying  out  by  keeping  bits  of 
stale  bread  in  cultures. 

Euglena  will  be  found  in  some  'of  the  cultures,  but  usually  not 
in  any  quantities  before  the  end  of  four  or  five  weeks.  They 
appear  along  the  side  of  the  dish  toward  the  light. 

Carchesium  and  Vorticella  are  frequently  found  on  decaying 
duckweed  (Lemna)  and  horn  wort  (Ceratophyllum).  To  secure  a 
culture,  have  a  more  plentiful  supply  of  water  than  for  ameba. 
Professor  Walton  tells  me  that  he  always  finds  a  supply  of 
Epistylis  on  the  shells  of  fresh  water  snails. 

215 


216  Animal  Micrology 

Opalina  may  be  obtained  readily  by  killing  a  frog  wich  chlo- 
roform and  slitting  open  the  large  intestine.  Examine  scrapings 
of  the  epithelial  wall  in  normal  saline  (reagent  74,  Appendix  B). 

Sporozoa.  Gaegarina  may  be  found  in  the  alimentary  canal  of 
the  cockroach  and  Monocystis,  in  the  male  reproductive  organs  of 
the  earthworm.  They  are  best  studied  in  normal  saline.  If  it  is 
desired  to  stain  and  mount  specimens  they  may  be  fixed  in  corro- 
sive-acetic (reagent  14,  Appendix  B)  for  5  minutes,  washed  thor- 
oughly in  35  per  cent,  alcohol  to  which  a  little  tincture  of  iodine 
has  been  added,  and  stained  with  Ehrlich's  triple  stain  (reagent 
39),  or  hematoxylin  and  acid  fuchsin  (reagents  49  and  42). 

b)  Quieting  infusoria. — 1.  Let  sufficient  water  evaporate  from 
under  the  cover  to  permit  the  latter  to  press  lightly  upon  the  ani- 
mals. Guard  against  too  great  evaporation  of  water  or  the  infu- 
soria will  be  crushed. 

2.  Entanglement  in  fibers  of  cotton,  etc.,  sometimes  proves 
efficacious. 

3.  A  small  amount  of  gelatin  or  better,  cherry-tree  gum,  dis- 
solved in  water  makes  a  viscous  mass  which  is  often  useful  in 
retarding  their  motions.  A  bit  of  white  of  egg  may  be  used  in 
the  same  way. 

4.  Animals  may  be  narcotized  by  means  of  a  small  drop  of 
very  dilute  alcohol  (preferably  methyl  alcohol)  or  chloretone 
(about  one  drop  of  a  1  per  cent,  solution  to  10  drops  of  water). 
(Chloretone  is  manufactured  by  Park,  Davis  &  Co.,  of  Detroit, 
Mich.  For  its  use  as  an  anaesthetic  in  biological  work  see  Jour- 
nal of  Applied  Microscopy,  Vol.  V,  p,  2051.) 

c)  Feeding. — Place  finely  pulverized  carmine  or  indigo  under 
the  cover-glass.  The  colored  powder  rapidly  accumulates  in  the 
food  vacuoles.  In  such  a  preparation  the  action  of  the  cilia  of 
infusoria  is  also  indicated  by  the  rapid  movement  of  the  particles 
in  the  vicinity  of  the  animal.     See  also  chap,  xiv,  memorandum  4. 

d)  Staining. — For  intra  vitam  staining  see  Appendix  B,  rea- 
gents 55a,  31,  and  58. 

To  see  cilia  of  infusoria  treat  the  animal  with  very  dilute 
iodine  solution  or  a  drop  of  a  dilute  solution  of  tannin. 


Appendix  D:  Preparation  of  Microscopical  Material     217 

To  see  the  macronucleus  and  the  micronucleus  use  a  drop  of  a 
2  per  cent,  solution  of  acetic  acid  or,  better,  methyl  green  (Appen- 
dix B,  reagent  56) . 

e)  Permanent  mounted  preparations. — Benedict's  method  is 
as  follows: 

"Smear  a  glass  slide  with  albumen  fixative,  as  in  preparing  for 
the  mounting  of  paraffin  sections.  Then  place  on  the  surface  of 
the  film  of  fixative  a  drop  or  two  of  water  containing  the  forms 
which  it  is  desired  to  stain.  Let  nearly  all  the  water  evaporate 
by  exposure  to  the  air  of  the  room  until  only  the  film  of  fixative 
remains  moist.  The  slide  can  now  be  immersed  in  Gilson  or  any 
other  fixing  reagent,  and  then  passed  through  the  alcohols,  stains, 
etc.,  in  the  same  way  that  mounted  sections  are  handled. 

"  I  have  had  no  difficulty  in  getting  preparations  of  Paramoe- 
cium  by  this  method,  with  very  little  distortion  of  the  body,  and 
any  kind  of  staining  desired.  By  this  method  students  can  pre- 
pare in  ten  minutes  very  satisfactory  preparations  of  protozoa  for 
demonstration  of  nuclei,  etc." — Journal  of  Applied  Microscopy, 
Vol.  VI,  p.  2647. 

SPONGES 

To  isolate  the  spicules  of  calcareous  sponges  boil  a  bit  of  the 
sponge  in  5  per  cent,  solution  of  caustic  potash  for  a  few  minutes. 

Fairly  thick  transverse,  longitudinal,  and  tangential  sections 
of  Grantia  showing  spicules  in  the  tissues  are  useful.  Make 
these  with  an  old  razor  or  sharp  scalpel.  To  hold  the  object 
while  sectioning,  place  it  between  two  pieces  of  pith  or  cork."  For 
a  careful  study  of  the  relations  of  the  two  systems  of  canals  in  the 
body-wall,  thinner  sections  are  necessary.  To  prepare  these  it  is 
best  to  decalcify  (2  per  cent,  chromic  acid,  24  to  36  hours)  the 
sponge  and  cut  celloidin  or  paraffin  sections  on  the  microtome 
although  fairly  good  sections  may  be  made  by  hand.  They 
should  be  dehydrated  and  mounted  in  balsam  if  permanent  prep- 
arations are  desired;  if  not,  they  may  be  examined  in  glycerin. 

To  color  the  collar  cells  use  an  aqueous  solution  of  anilin  blue. 

Spicules  of   silicious  sponges  are  isolated  by  treating  bits  of 


218  Animal  Micrology 

the  sponge  with  strong  nitric  acid  or  a   mixture  of  nitric  and 
hydrochloric  acid. 

.  COELENTERATES 

Hydra  should  be  sought  for  in  spring-fed  pools.  In  the 
autumn  they  are  found  most  frequently  on  smooth  dead  leaves 
which  are  completely  submerged.  Material  should  be  collected 
and  placed  in  battery  jars  or  larger  glass  jars,  which  are  then 
filled  with  fresh,  clear  water  and  placed  in  a  fairly  light  place,  but 
not  too  near  a  window.  Put  a  small  amount  of  horn  wort  or  Chara 
in  each  jar.  In  a  few  hours  ( 12-36)  the  hydra  will  be  found  attached 
to  the  sides  of  the  vessel  and  to  the  plants.  They  may  readily  be 
kept  in  the  laboratory  throughout  the  winter  if  glass  plates  are 
placed  over  the  jars  to  prevent  excessive  evaporation  and  the 
temperature  is  not  allowed  to  go  below  freezing.  Fresh  water 
should  be  added  from  time  to  time  to  make  up  for  evaporation. 
In  case  their  supply  of  food  (Cyclops,  Daphnia,  and  other  small 
Crustacea)  is  exhausted  it  should  be  renewed  by  skimming  out 
from  other  aquaria  the  small  forms  upon  which  the  animal  feeds 
and  putting  them  in  the  hydra  jars. 

For  staining  and  mounting  entire  see  chap,  xiii  II,  B.  Kill  in 
the  same  way  for  sectioning.  The  most  instructive  sections  are 
(1)  transverse  sections,  (2)  longitudinal  sections  through  the 
mouth  and  a  bud,  and  (3)  sections  showing  the  sexual  organs. 
Stain  in  bulk  with  hematoxylin  (reagent  49,  Appendix  B),  imbed 
in  paraffin  using  the  method  for  delicate  objects  (chap,  vi,  VII), 
and  after  the  paraffin  has  been  removed  from  the  sections,  stain 
them  for  a  few  seconds  in  acid  fuchsin.  Dehydrate  and  mount  in 
the  usual  way. 

The  sections  are  much  more  satisfactory  if  the  hydra  have  been 
placed  in  small  stender  dishes  filled  with  filtered  water  (not  dis- 
tilled) and  kept  from  food  for  a  week  or  ten  days  before  killing. 
This  eliminates  the  metabolic  products  and  oil  globules  which 
ordinarily  obscure  the  details  of  structure. 

To  Stain  the  Nematocysts  of  Living  Hydra,  place  several  of  the 
animals  in  a  small  stender  dish  of  water  which  has  been   tinted  a 


Appendix  D:  Preparation  of  Microscopical  Material     219 

sky  blue  through  the  addition  of  methylen  blue  solution  made  up 
as  follows: 

Methylen  blue 1.0  gram 

Castile  soap 0.5  gram 

Water 300.0     c.c. 

After  two  hours  the  hydra  may  be  transferred  to  fresh  water;  the 
nematocyst  cells  are  stained  a  deep  blue.  (Method  of  Little, 
Journal  of  Applied  Microscopy,  Vol.  VI,  p.  2116.) 

To  Discharge  Nematocysts  drum  on  the  cover-glass  gently  with 
a  pencil.  By  using  a  very  small  opening  to  the  diaphragm  they 
are  usually  sufficiently  distinct  without  staining. 

For  Other  Polypoid  Forms,  the  methods  given  for  hydra  will 
answer  in  most  cases. 

For  Collecting  Free-Swimming  Medusoid  Forms  full  directions 
will  be  found  in  Brook's  Invertebrate  Zoology. 

Compound  Hydrozoa  should  be  placed  alive  into  the  cells  which 
they  are  to  occupy  when  mounted.  One  per  cent,  formic  acid  is 
then  added  drop  by  drop  to  the  sea-water.  After  the  animals 
have  been  killed,  the  fluid  is  replaced  by  glycerin-jelly  and  the 
cover-glass  is  put  in  place.  Another  method  is  to  kill  the  animals 
slowly  by  adding  a  few  crystals  of  chloral  hydrate,  from  time  to 
time,  to  the  small  vessel  of  sea-water  containing  them. 

Small  Jelly-Fish  may  be  fixed  and  hardened  in  1  per  cent, 
osmic  acid  and,  stained  or  unstained,  mounted  in  cells. 

PLAN ARIA 

Look  for  planarians  on  the  under  sides  of  stones  in  small 
streams  of  running  water.  They  are  usually  examined  alive.  To 
see  them  thrust  out  the  proboscis,  keep  them  from  food  for  a  few 
days  and  then  feed  them  on  dead  flies.  Planaria  which  have  been 
kept  in  the  laboratory  for  months  display  the  internal  organs 
much  more  clearly  than  freshly  captured  ones. 

If  it  is  desired  to  study  stained  specimens,  for  preparation  see* 
chap,  xiii,  iv,  A. 

To  Kill  Planaria  with  Pharynx  Protruded  Cole  [Journal  of  Ap- 
plied   Microscopy,    Vol.    VI,    p.    2125)    recommends    covering 


220  Animal  Micrology 

them  in  a  watch-glass  with  a  1  per  cent,  aqueous  solution  of 
chloretone  until  they  are  immobilized  and  then  rapidly  transfer- 
ring them  to  5  per  cent,  formalin.  Other  fixing  agents  than 
formalin  can  be  used. 

DISTOMES 

Perhaps  the  most  easily  obtained  form  is  the  one  which  is 
found  in  the  liver  of  the  cat.  Search  for  it  in  the  bile  passages. 
Fix  it  in  hot  corrosive  sublimate,  wash  out  with  alcohol  to  which 
tincture  of  iodine  has  been  added,  and  stain  for  24  hours  in  alum 
cochineal  (reagent  27,  Appendix  B),  or  hemalum  (reagent  47). 
As  with  Planaria,  they  should  be  compressed  between  two  glass 
slides  (see  chap,  xiii,  iv,  A,  7). 

If  the  large  liver  fluke  of  the  sheep  (Fasciola  hepatica)  can 
be  obtained,  both  the  alimentary  canal  and  the  excretory  system 
may  be  injected  with  finely  powdered  carmine  in  water.  A  sepa- 
rate fluke  should  be  used  in  each  case.  For  injection,  a  very  fine- 
pointed  cannula  with  rubber  cap  is  used,  or  the  manipulator  may 
operate  the  cannula  by  simply  blowing  through  it.  The  excretory 
system  is  injected  through  an  incision  made  with  a  sharp-pointed 
scalpel  in  the  median  line  near  the  hinder  end  of  the  animal.  For 
the  alimentary  canal,  the  incision  should  be  made  about  1  mm.  to 
one  side  of  the  median  line.  When  the  injection  is  completed, 
flatten  the  animal  somewhat  between  two  slides  (see  chap,  xiii, 
iv,  A,  7),  harden  in  95  per  cent,  alcohol  for  12  to  24  hours,  then 
dehydrate,  clear,  and  mount  in  balsam. 

CESTODES 

Near  large  cities  an  unlimited  supply  of  the  sheep  tapeworm 
(Monieza)  can  usually  be  secured  from  slaughter  houses.  Ample 
supplies  can  ordinarily  be  obtained  from  dogs,  or,  less  frequently, 
from  cats.  Tapeworms  can  be  kept  alive  for  considerable  length 
of  time  in  tepid  normal  saline.  The  most  instructive  portions  to 
mount  are  scolex,  and  sexually  mature  proglottids.  For  fixing 
and  staining  use  the  same  methods  as  for  distomes  with  the  excep- 
tion that  cold  instead  of  hot  corrosive  sublimate  should  be  used. 
The  scolex  should  not  be  compressed. 


Appendix  D:  Preparation  of  Microscopical  Material     221 

To  Find  Cysticerci,  open  the  body  cavity  of  a  rabbit  and  look 
for  large  whitish  bodies  imbedded  in  the  peritoneum  or  liver  (the 
cysticercus  of  T.  serrata).     Likewise,  the  cysticercus  of  T.  cras- 


Fig.  70. — Compressor. 

sicollis  may  be  found  in  the  liver  of  the  mouse.  If  a  cysticercus 
is  found,  its  outer  wall  should  be  slit  open  in  order  to  show  the 
reversed  scolex. 

ASCARIS 
See  chap,  xvi,  memorandum  11. 

TRICHINA 

The  simplest  way  to  obtain  it  is  to  apply  for  infected  pork 
to  the  government  inspector  whose  headquarters  are  to  be  found 
near  all  large  slaughter  houses   in  cities.       Bits  of  the  infected 


Fig.  71. — Compressor  Used  by  the  Government  Bureaus  for  Meat  Inspection. 

muscle  should  be  teased  and  flattened  out  in  a  compressor  (Figs. 
70  and  71)  until  a  favorable  area  has  been  found.  The  flattened 
tissue  may  then  be  dehydrated  and  mounted  unstained  or  it  may  be 
stained  in  hematoxylin  (reagent  49,  appendix  B).  Better  results 
will  be  obtained  if  the  material  is  fixed  for  from  4  to   6   hours  in 


222  Animal  Micrology 

Carnoy's  fluid  (reagent    2)    before  dehydrating  or  staining.     If 
desired,  the  tissue  may  be  sectioned  in  celloidin  or  paraffin. 

To  Demonstrate  Living  Trichinae  Barnes  {American  Monthly 
Microscopical  Journal,  Vol.  XIV,  p.  104)  subjects  small  bits  of 
trichinized  muscle  to  a  mixture  of  3  grains  of  pepsin,  2  drams  of 
water,  and  2  minims  of  hydrochloric  acid,  for  about  three  hours  at 
body  temperature  with  occasional  shaking.  When  the  flesh  and 
cysts  are  dissolved,  the  liquid  is  poured  into  a  narrow  glass 
vessel  and  allowed  to  settle.  The  live  trichinae  may  be  withdrawn 
with  a  pipette  from  the  bottom  of  the  fluid  and  examined  on  a 
warm  stage. 

ROTIFERS 

Rotifers  will  usually  be  found  in  abundance  in  some  of  the 
laboratory  aquaria  on  the  lighted  side  of  the  vessel.  For  ordinary 
class  work  they  are  best  studied  alive.  They  are  difficult  to 
preserve  properly.  Full  directions  for  killing  and  preserving 
will  be  found  in  Jenning's  paper,  "Rotatoria  of  the  United  States," 
U.  S.  Fish  Commission  Bulletin,  1902,  p.  277. 

To  Quiet  Rotifers,  Cole  {Journal  of  Applied  Microscopy,  Vol. 
VI,  p.  2179)  anaesthetizes  them  by  adding  from  time  to  time  a 
drop  of  1  per  cent,  aqueous  solution  of  chloretone  to  the  water  on 
the  slide  in  which  the  animals  are  being  examined. 

BRYOZOA 

They  may  be  treated  in  the  same  way  as  compound  hydrozoa. 
Plumatella  may  frequently  be  found  in  shallow  fresh-water 
streams  on  the  under  side  of  flat  rocks;  Pectinatella,  in  rivers 
and  streams  on  the  upper  surface  of  mussel  shells,  etc. 

EARTHWORM 
Earthworms  are  best  collected  on  warm,  rainy  nights  when 
they  may  be  found  extended  on  the  surface  of  the  ground  near 
their  burrows.  They  are  most  plentiful  in  old  gardens  or  rich 
lawns.  A  lantern  and  a  pail  are  the  only  implements  necessary. 
Earthworms  may  frequently  be  found,  however,  in  large  numbers 
on  the  surface  of  the  ground  on  cloudy  days  immediately  after 
prolonged  hard  rain. 


Appendix  D :  Preparation  of  Microscopical  Material     223 

To  Prepare  Earthworms  for  Sectioning,  secure  good-sized  speci- 
mens, wash  them  in  water  and  place  them  in  a  vessel  containing 
moist  filter  paper.  There  must  not  be  sufficient  water  to  drown 
the  worms  nor  little  enough  to  let  the  paper  dry  out.  Put  only 
a  few  worms  in  each  dish  and  adjust  the  cover  so  as  to  admit  a 
little  air.  After  12  to  24  hours  it  is  well  to  remove  any  dead  or 
injuried  specimens  and  to  change  the  filter  paper.  The  dish  should 
be  kept  from  direct  sunlight  in  a  cool  place.  After  two  or  three 
days  the  grit  and  dirt  in  the  alimentary  canal  will  have  been 
passed  out  and  its  place  taken  by  paper  which  the  worms  have 
eaten.     They  are  then  ready  to  kill  and  preserve  or  section. 

Place  the  worms  in  a  flat  vessel  and  pour  on  sufficient  water 
to  cover  them.  During  the  next  two  hours  add  a  little  alcohol 
from  time  to  time  until  the  strength  of  the  liquid  is  increased  to 
about  8  or  10  per  cent.  Then  wash  all  mucous  from  the  body  of 
the  worms  and  replace  them  in  10  per  cent,  alcohol  until  they  no 
longer  respond  to  pricking  or  pinching  with  forceps.  Transfer 
them  to  50  per  cent,  alcohol  for  several  hours,  keeping  them 
straightened  out  as  much  as  possible;  then  to  70  per  cent,  alcohol 
for  12  hours,  followed  by  95  per  cent,  alcohol  for  24  hours. 
Preserve  finally  in  70  per  cent,  alcohol. 

For  sectioning  the  preliminary  steps  are  the  same  but  from 
10  per  cent,  alcohol  the  worms  should  be  placed  into  Zenker's 
fluid  (reagent  6,  Appendix  B)  for  4  to  6  hours.  For  washing, 
etc.,  follow  the  directions  given  in  the  discussion  of  the  reagent. 
To  facilitate  penetration  of  the  fluid,  it  is  well  to  slit  open  the 
body  cavity  of  the  worm  in  places  that  are  not  to  be  sectioned. 
The  most  instructive  sections  are  cross-sections  of  the  middle  of 
the  body,  and  sagittal  sections  of  the  anterior  end  which  include 
the  pharynx.  The  worms  may  be  stained  in  bulk  (24  to  36 
hours)  in  borax-carmine  (reagent  3^)  or  hematoxylin  (reagent 
49)  before  sectioning. 

Entire  nephridia  together  with  a  small  part  of  the  septum 
which  they  traverse  should  be  carefully  dissected  out,  stained  in 
borax-carmine  (reagent  32),  dehydrated,  cleared,  and  mounted 
in  balsam. 


224  Animal  Micrology 

An  ovary  should  be  removed  entire,  stained  with  borax-car- 
mine, dehydrated,  cleared,  and  mounted  in  balsam. 

A  testis  should  be  treated  in  the  same  way  as  an  ovary.  Tease 
it  in  the  balsam  before  adding  the  cover-glass. 

To  Keep  Earthworms  Alive  in  Winter,  Jennings  {Journal  of 
Applied  Microscopy,  Vol.  VI,  p.  2412)  places  them,  immediately 
after  collection,  into  bacteria  dishes  (9  in.  in  diameter  by  3  in. 
deep)  between  folds  of  muslin  which  is  kept  damp  but  not  drip- 
ping wet.  Not  more  than  a  dozen  worms  should  be  placed  in  one 
dish  and  the  cloth  should  be  changed  or  washed  at  least  every 
two  weeks.  The  worms  may  be  fed  on  leaves,  etc.,  from  time  to 
time. 

To  Immobilize  Earthworms  for  study  of  circulation  of  the  blood 
under  the  microscope  or  projection  lantern,  Cole  {Journal  of 
Applied  Microscopy,  Vol.  VI,  p.  2125)  places  them  in  a  0.2 
per  cent,  aqueous  solution  of  chloretone  for  3  or  4  minutes.  Such 
worms  may  be  slightly  compressed  between  two  slides. 

To  Examine  Corpuscles  of  the  Coelomic  Fluid,  expose  the  worms 
for  a  minute  or  two,  to  the  vapor  of  chloroform.  The  coelomic 
fluid  exudes  through  dorsal  pores..  Touch  a  cover-glass  to  the 
fluid  and  mount. 

The  Setae  Can  Be  Isolated  by  boiling  a  bit  of  the  tissue  con- 
taining them  in  a  solution  of  caustic  potash.  When  isolated,  dry 
them  and  mount  in  balsam. 

LEECH 

Leeches  are  obtained  from  fresh-water  pools,  streams  and 
marshes,  but  to  get  sufficient  numbers  for  class  use  it  is  usually 
necessary  to  purchase  them  from  dealers.  Live  leeches  intended 
for  dissection  may  be  killed  with  chloroform.  Cross-sections 
prepared  in  the  same  way  as  for  earthworms  are  very  instructive. 

ARTHROPODS 
For  Mounting  Small  Crustacea  see  chap,  xiii,  iii,  A. 
To  Quiet  Small  Crustacea  for  Microscopical  Examination  (Cole: 
Journal  of  Applied  Microscopy,  Vol.   VI,  p.  2180)  place  them 
in  a  watch-glass  containing  2  parts  of  1  per  cent,  chloretone  and 


Appendix  D :  Preparation  of  Microscopical  Material     225 

5  parts  of  water.  The  same  treatment  is  useful  for  the  larvae  of 
insects.  Some  such  as  the  nymph  of  the  dragon-fly,  will  require 
more  chloretone. 

For  Various  Dissections  and  Parts  of  Insects  see  chap,  x,  ii. 

For  Mounting  Insects  Entire  (beetles,  mosquitoes,  gnats,  aphids, 
larvae,  etc. )  as  microscopic  preparations,  and  for  mounting  muscle, 
wings,  heads,  legs,  scales,  antennae,  etc.,  see  chap.  xiii. 

Live  nymphs  of  the  dragon-fly  are  especially  valuable  for  study 
under  the  compound  microscope  because  they  show  very  clearly 
the  valvular  action  of  the  heart,  the  tracheal  gills  and  tracheae, 
and  the  brain  and  its  relation  to  the  eyes.  The  heart  is  located 
well  toward  the  posterior  end  of  the  abdomen  between  the  main 
tracheal  trunks.  Cole  [Journal  of  Applied  Microscopy,  Vol. 
VI,  p.  2274)  recommends  that  the  animals  be  anaesthetized  by 
subjecting  them  to  a  1  per  cent,  aqueous  solution  of  chloretone. 

MOLLUSKS 

Gills  of  the  Fresh-Water  Mussel  may  be  fixed  in  corrosive  sub- 
limate (reagent  13,  Appendix  B)  for  from  20  to  30  minutes, 
washed  out  in  water  and  then  in  dilute  alcohol  to  which  tincture 
of  iodine  has  been  added.  Make  cross-sections  in  paraffin,  stain 
in  dilute  hematoxylin  (reagent  49)  and  mount  in  the  ordinary  way. 

Cross-Sections  of  the  Entire  Mussel  are  valuable  to  show  the 
relations  of  the  gills,  kidneys,  and  heart.  Wedge  the  valves  apart 
slightly  and  immerse  the  animal  for  24  hours  in  1  per  cent, 
chromic  acid  (reagent  10).  Wash  out  thoroughly  in  running 
water  and  transfer  the  specimens  to  70  per  cent,  alcohol  for  two 
or  three  days  or  until  needed.  To  section,  remove  both  valves, 
place  the  animal  on  a  board  and  with  a  razor  cut  transverse  sec- 
tions. These  are  to  be  examined  with  the  naked  eye  or  with  a 
dissecting  lens. 

To  Kill  Snails  in  an  Expanded  Condition  put  them  into  a  vessel 
of  cold  water,  then  run  a  layer  of  hot  water  onto  the  surface  of 
the  cold  water.  See  that  the  vessel  is  full  of  water  and  cover  it 
with  a  glass  plate  to  exclude  the  air. 

For  Lingual  Ribbon  of  the  Snail  see  chap,  xiii,  memorandum  7. 


226  Animal  Micrology 

AMPHIOXUS 

Specimens  must  ordinarily  be  secured  from  dealers.  The  ani- 
mals should  be  stained  entire  in  borax-carmine  (reagent  32, 
Appendix  B)  and  sectioned  in  celloidin.  The  most  instructive 
sections  are  cross-sections  of  a  female  with  well  developed  gonads, 
and  longitudinal  sections  of  small  individuals.  Mounts  of  entire 
small  specimens  should  also  be  made. 

VERTEBRATA 

For  any  of  the  tissues  of  vertebrates  which  teachers  may  desire 
to  prepare,  ample  directions  are  given  in  Appendix  C. 

For  Demonstration  of  Circulation  of  the  Blood  in  the  frog,  see 
chap.  xiv. 


APPENDIX  E 
TABLE  OF  EQUIVALENT  WEIGHTS  AND  MEASURES 

WEIGHTS,  METRIC  AND  AVOIRDUPOIS 

1  kilo=l,000  grams=l  liter  of  water  at  its  maximum  density=2.2  pounds. 
1  gram=l  cubic  centimeter  of  water  at  its  maximum  density =15.43 

grains =0.035  ounce. 
1  pound=453.59  grams. 
1  ounce=28.35  grams. 
1  grain  (Troy)=0.065  gram. 
1  dram=1.77  grams. 

WEIGHTS,  METRIC  AND  APOTHECARIES' 

1  kilo=l,000  grams. 

1  gram =15.43  grains =0.032  ounv  . 

1  pound=373.24  grams. 

1  ounce =31. 10  grams. 

1  dram =3.89  grams. 

1  scruple =1.30  grams. 

1  grain=0.065  gram. 

MEASURES  OF  LENGTH,  METRIC  AND  ENGLISH 

1  meter= 1,000  millimeters =39.37  inches. 

1  centimeter=0.394  inch. 

1  millimeter =0.039  inch. 

1  yard=0.914  meter. 

1  foot=30.48  centimeters. 

1  inch=2.54centimeters=25.40  millimeters. 

LIQUID  MEASURES,  METRIC  AND  APOTHECARIES' 

1  liter=l,000  cubic  centimeters =2. 11  pints. 
1  cubic  centimeter =0.034  fluid  ounce= 16.23  minims. 
1  gallon=128  ounces =3.79  liters. 
1  pint=16  ounces=473.18  cubic  centimeters. 
1  fluid  ounce=8  fluid  drams=29.57  cubic  centimeters. 
1  fluid  dram=60  minims=3.70  cubic  centimeters. 

227 


228  Animal  Micrology 

THERMOMETERS 

To  reduce  degrees  Fahrenheit  to  degrees  Centigrade  use  the  formula, 
C=5/9  (F — 32).  For  example,  if  the  number  of  degrees  Fahrenheit  is  77, 
then  C=5/9  (77— 32)=25  degrees.  Or,  for  instance,  to  reduce  —31  degrees 
Fahrenheit  to  Centigrade,  C=5/9(-31-32)=5/9x -63= -35  degrees. 

To  reduce  degrees  of  Centigrade  to  degrees  of  Fahrenheit  use  the 
formula  F=9/5  C+32.  For  example,  if  the  number  of  degrees  Centi- 
grade is  25,  then  F=(9/5x25)+32=77  degrees.  Or,  to  reduce  -35 
degrees  Centigrade  to  Fahrenheit,  F=(9/5x  -35)+32=-31  degrees. 


INDEX 


INDEX 


Abbe,  156  ;  camera  lucida,  147. 

Abbot's  method  for  staining  spores  of  bac- 
teria, 111. 

Aberration,  spherical,  138 ;  correction  of,  139. 

Absolute  alcohol,  testing  for  water,  56. 

Absorption  of  fat,  198. 

Accessory  chromosome,  191. 

Acetic  acid,  161. 

Acetic  alcohol,  161 ;  and  chloroform,  123, 125, 
161. 

Achromatic  objective,  145. 

Achromatism,  145. 

Acid  fuchsin,  seefuchsin. 

Acidophil  granules,  190. 

Action  of  liquids,  to  hasten,  56. 

Adenoid  connective  tissue,  197. 

Adipose  tissue,  194. 

Affixing  sections,  23,  41,  64. 

Air  bubbles,  159, 160. 

Albumen  fixative,  12,  23,  41. 

Albumenoids,  15. 

Albuminous  coats  of  eggs,  to  remove,  118. 

Alcohol,  absolute,  7, 13,  49,  84;  acid,  8,  24,  50 
162;  alkaline,  49,  50;  and  chloroform  162 
ether,  8,  23;  ethyl,  13;  fixation,  28,  162 
methyl,  13;  replenishing,  56. 

Alcoholometer,  13. 

Alimentary  canal,  197, 198;  of  cockroach,  77. 

Alum  carmine,  171, 181 ;  dahlia,  191. 

Alum  cochineal,  126, 170, 181. 

Aluminium  chloride,  184. 

Altmann,  192 

Ameba,  215. 

Ameboid  movement  in  leucocytes,  102. 

Amphibia,  material  for  the  embryology  of, 
118;  fecundation  and  early  embryonic 
stages,  122 ;  to  study  eggs  of,  118. 

Amphibian  eggs,  167. 

Amphioxus,  226. 

Amphophil  granules,  190. 

Amitosis,  191. 

Ammonia  copper  sulphate  solution,  152. 

Ammonium  chromate,  214. 

Ammonium  picrate,  181, 183. 

Amyloid,  172, 199. 

Anatomical  specimens,  to  preserve,  31. 

Andrews,  117. 

Angular  aperture,  145. 

Anilin  blue,  217 ;  and  orange  G,  172. 

Anilin,  alcohol,  182;  dyes,  19,  20,  57,  63,  171; 
formulae,  171 ;  water,  171. 

Antennae  of  insects,  93. 

Anthrax,  109,  111. 

Aorta,  193. 


Apertometer,  145. 

Aphid,  91. 

Aplanatism,  145. 

Apochromatic  objective,  146. 

Apparatus,  1 ;  dealers  in,  5. 

Appliances,  microscopical,  145. 

Aqueous  humor,  187. 

Arcella,  215. 

Areas  of  Conheim,  204. 

Areolar  tissue,  194. 

Arrangement  of  apparatus  and  reagents,  6, 

49. 
Artery,  193. 
Arthropods,  224. 
Ascaris,  162;  maturation,  fertilization,  and 

cleavage,  123. 
Asphalt-paraffin-rubber   method  of  imbed- 
•     ding,  44, 119. 
Aurantia,  20. 
Axial  illumination,  150. 
Axis  cylinder,  206. 
Axone,  206. 

Bacillus,  108;  aerogenes  capsulatus,  111;  of 
anthrax,  109,  111;  of  bubonic  plague,  111; 
of  chancroid,  111 ;  coli  communis,  111 ;  diph- 
theriae,  109,  111 ;  of  dysentery,  111 ;  of  glan- 
ders, 111;  of  influenza,  111;  of  malignant 
edema,  111 ;  mucosus  capsulatus,  111 ;  pro- 
teus,  111;  pyanocyaneus,  111;  of  tetanus, 
111 ;  of  tuberculosis,  111 ;  of  typhoid,  111. 

Bacteria,  cover-glass  preparations  of,  105; 
features  to  be  observed  in  studying,  108; 
Gram's  method  of  staining,  107 ;  hanging- 
drop  preparations  of,  105,  108;  m  tissues, 
105,  107;  material  for  demonstrating,  108; 
methylen  blue  stain  for,  107;  mounting 
from  fluid  media,  105;  mounting  from 
solid  media,  105;  staining  and  mounting 
films,  105;  spores,  108;  staining  flagella, 
111 ;  stains  for,  109. 

Bacterial  examination,  105. 

Balsam,  11,  22,  34, 80, 90;  bottle,  4;  exudation, 
to  remove,  58. 

Bardeen,  128, 129;  microtome,  67. 

Basophil  granules,  190. 

Basophil  substance  in  nerve-cells,  182. 

Bausch,  152, 160. 

Bausch  and  Lomb  microscope,  143. 

Beale's  carmine,  173. 

Bee,  93. 

Beetle,  92. 

Beggiatoa,  108. 

Bell's  cement,  79. 

Benedict,  217. 

Benzopurpurin,  20. 

Berlin  blue,  81. 


231 


232 


Animal  Micrology 


Bethe's  fluid,  181. 

Bichloride  of  mercury,  165;  see  also  corrosive 
sublimate. 

Bichromate  of  potassium,  162, 187;  and  acetic 
acid,  163;  and  corrosive  sublimate,  163; 
and  cupric  sulphate,  163;  and  sodium 
sulphate,  163. 

Binocular  microscope,  146. 

Bioblasts,  192. 

Bismarck  brown,  20, 106, 173. 

Bladder,  213. 

Blastoderm  of  chick,  113, 114. 

Blastoderms,  to  orient  colorless,  117. 

Bleaching,  24,  45. 

Bleu  de  Lyon,  20 ;  see  also  Lyons  blue. 

Blocks  for  celloidin  imbedding,  59,  62. 

Blood,  97, 190 ;  cover-glass  preparations,  98 
clinical  examination  of,  99;  corpuscles 
172,  176;  corpuscles,  living,  97;  crystals 
97, 190 ;  currents,  to  observe.  101 ;  dry  prep 
arations  of,  98;  effects  or  reagents  on 
97 ;  enumeration  of  corpuscles,  99 ;  exami 
nation  of  fresh,  97;  platelets,  97;  rapid 
method,  99;  serum,  187;  Loeffler's  serum, 
110 ;  Schultze's  iodized  serum,  187 ;  to  study 
in  sections,  101;  test  for,  98;  Wright's 
stain  for,  102. 

Blood  forming  organs,  190. 

Blow  pipe,  1. 

Bone,  79;  corpuscles  and  their  processes, 
194;  decalcified,  79,  189,  194;  endochondral 
development  of,  194;  fibers  of  Sharpey, 
195;  grinding,  80;  Haversian  canals  and 
lamellae,  195;  intra-membranous  devel- 
opment of,  195 ;  isolation  of  corpuscles  and 
of  lamellae,  195;  sectioning,  79,  80;  young, 
to  decalcify,  189. 

Borax-carmine,  9,  49,  51,  54,  61,  77,  89,  91, 119, 
121, 169;  formula,  9. 

Bordeaux  red,  10,  49,  53,  55, 117, 173;  formula, 
10. 

Born,  127. 

Bottle-capping,  30. 

Bouillon,  glucose,  110. 

Box,  slide,  1. 

Brain,  206,  207. 

Brain  cells,  206. 

Brain  sand,  206. 

Brittle  objects,  sectioning,  43,  44,  63. 

Bronchi,  211. 

Brownian  movement,  146. 

Brushes,  camel's  hair,  1. 

Bryozoa,  222. 

Bubonic  plague,  bacillus  of,  111. 

Bull's  eye,  151. 

Bunge-Loeffler,  staining  flagella  of  bacteria, 
111. 

Btitschli,  192. 

Butterfly,  wings  of,  92 ;  eggs  of,  93. 

Calendar,  1 ;  record,  6. 
Calleja's  staining  fluid,  174. 
Camel's  hair  brushes,  1. 
Camera,  photographic,  155. 
Camera  lucida,  146, 147, 148. 
Canada  balsam,  11. 


Cannulae,  82,  85. 

Capillaries,  blood,  193;  lymph,  193. 
Carbol-fuchsin  for  bacteria,  109. 
Carbolic  acid,  21,  22. 
Carbol-xylol  clearer,  9,  21,  34. 
Carbon  dioxide,  for  freezing,  67 ;  for  killing, 
16. 

Carchesium,  215. 

Card  records,  1,  6. 

Carmalum  ;76 :  formula,  173. 

Carmine,  19,  57,  162;  acid,  76,  123, 174;  alum, 
168;  and  Lyons  blue,  51 ;  Beale's,  173 ;  injec- 
tion mass,  81 ;  picric  acid  and  indigo  car- 
mine, 174 ;  picro-,  10, 184. 

Carnoy,  161. 

Carpenter  and  Dallinger,  160. 

Carrier  for  paraffin  ribbon,  41. 

Cartilage,  195;  capsule  of,  195;  connective 
tissue  and  elastic  fibers  in,  195;  elastic 
(yellow-fibro-),  195;  glycogen  in,  195;  hya- 
line, 196;  white  fibro-,  196. 

Caustic  potash,  187. 

Cell,  animal,  191;  living  or  fresh.  192;  of 
Paneth,  197;  pigment,  193;  prickle,  213; 
reduction  division  in,  193. 

Cell-making,  87,  95. 

Celloidin,  12,  22;  bottle,  4;  clearing  before 
sectioning,  64;  hardening,  61,  62;  imbed- 
ding, 59 ;  Gilson's  rapid  method,  64 ;  method, 
26,  59 ;  preparation  of  material  for  imbed- 
ding in,  53;  sections,  to  transfer  from  the 
knife,  64 ;  time  required  for  the  method,  62. 

Celloidin  and  paraffin  methods  compared, 
63. 

Celloidin-paraffin  infiltration,  63. 
Cells,  to  lessen  evaporation  from,  95. 
Cement,  Bell's,  79,  89. 
Cement  substance,  188. 
Center  of  slide,  to  find,  43. 
Centering  an  object  in  a  cell,  93. 
Centigrade  to  Fahrenheit  scale,  228. 
Central  illumination,  150. 
Central  nervous  system,  188,  206. 
Centrosome,  53. 
Cerebellar  cortex,  206. 
Cerebral  cortex,  206. 
Ceruminous  glands,  201. 
Cestodes,  220. 

Chancroid,  bacillus  of,  111. 
Chick,  embryology  of,  113;  stages  necessary 
for  a  course  in,  115, 116. 

Chick  embryos,  fixing,  staining,  and  mount- 
ing, 114, 115;  freeing  blastoderm  from  yolk 
in,  117;  orienting,  114}  116;  reconstruction 
of  heart,  126;  removing  blastoderm,  114; 
sectioning,  116. 

Child,  117, 118. 

Chironomous  larva,  gland  cells  of,  192. 

Chloretone,  216. 

Chloride  and  acetate  of  copper,  164. 

Cholera,  spirillum  of,  111. 

Choroid,  203. 

Choroid  plexus,  206. 

Chromatic  aberration,  139. 


Index 


233 


Chromic  acid,  118, 164, 189,  225;  and  its  com- 
pounds, 17,  164, 165. 

Chromic  acid  material,  bleaching,  45. 

Chromo-aceto-osmic  acid,  164. 

Chromo-platinic  mixture,  165. 

Chromosome,  53. 

Chrom-silver  method,  71. 

Cicatricula,  113. 

Cilia  of  infusoria,  216. 

Ciliated  epithelium,  75. 

Circulation,  in  foot  of  frog,  101 ;  in  mesen- 
tery, 101. 

Circulatory  system,  193. 

Citric  acid,  177. 

Cleaning  lenses,  158, 159, 160. 

Cleaning  slides  and  covers,  57. 

Cleanliness,  6,  44. 

Clearer,  21,  62;  carbol-xylol,  9,  21,  30;  Eycle- 
shymer's,  22,  62. 

Clearing,  21. 

Cleavage,  in  Ascaris,  123;  in  echinoderms, 
amphibia,  and  teleosts,  122;  in  living 
material  (snails),  123;  iu  mammals,  121. 

Clinging  of  paraffin  sections  to  the  knife,  48. 

Clitoris,  210. 

Clove  oil  for  minute  dissections,  78. 

Coal-tar  dyes,  20. 

Coccus,  108. 

Cochlea,  201 ;  nerve  fibers  and  nerve  endings 
of,  201. 

Cole,  219,  222,  224,  225. 

Collar  cells  of  sponge,  217. 

Collodion,  23,  44,  64. 

Colostrum,  213. 

Columnar  epithelium,  75. 

Compensating  ocular,  145, 148. 

Compressor,  221. 

Concave  or  diverging  lens,  134. 

Condenser,  148, 151. 

Conheim,  areas  of,  204. 

Conjugate  foci,  134. 

Conklin,  117, 122, 170;  picro-hematoxylin,  177. 

Connective  tissue,  172, 174, 185, 194,  196. 

Contractile  animals,  16, 161. 

Conventional  distance  of  vision,  144. 

Convex  or  converging  lens,  134. 

Coplin  staining  jars,  4,  49. 

Copper  sulphate  method  of  preparing  abso- 
lute alcohol,  7. 

Cornea,  203. 

Corneal  corpuscles  and  nerves,  203;  spaces 
and  canahculi,  2U3. 

Corpora  lutea,  119,  210. 

Correction  collar,  148. 

Corrosion,  24,  86. 

Corrosive  sublimate.  9, 18, 165 ;  handling,  166 ; 
and  acetic  acid,  166 ;  and  nitric  acid,  166. 

Cover-glass,  1 ;  supports  for,  78;  forceps,  106; 
correction,  148;  to  clean,  57. 

Creasote,  beechwood,  22.  62. 
Crescents  of  Gianuzzi,  198. 
Crooked  paraffin  ribbons,  46. 


Crown  glass,  140. 

Crumbling  of  tissues  in  paraffin,  43,  44,  46, 

47,  66. 
Crustacea,  small,  90,  224. 
Crystals,  blood,  97. 
Culture  slide,  108. 
Curare,  101. 
Cyanin,  211. 
Cyclops,  90,  218. 
Cylindrical  end  bulbs,  206. 
Cypris,  90. 
Cysticerci,  221. 
Cytological  work,  reagents  for,  162, 164, 170, 

175. 
Cytoplasmic  granules,  183. 

Damar,  22. 

Daphnia,  90,  218. 

Dealcoholization,  22. 

Dealers  and  manufacturers,  5. 

Decalcification,  24,  79 ;  fixing  before,  80. 

Decalcifying  fluid,  9,  79 ;  formulae,  189. 

Defective  mounts,  57,  58. 

Definition,  148. 

Dehydration,  18. 

Delicate  tissues,  43;  paraffin  method  for,  54; 

dehydrating  apparatus  for,  55;    freezing 

method  for,  70. 
Demilunes  of  Heidenhain,  198. 
Descemet,  membrane  of,  204. 
Desilicidation,  24. 
Desk,  4, 157. 
Desmids,  94. 

Determination  of  elements  that  will  be  im- 
pregnated in  Golgi  method,  73. 
Diamond,  writing,  1. 
Diapedesis,  101. 
Diaphragms,  149, 159. 
Difficulties    in    sectioning    paraffin,   table 

of,  46. 
Difflugia,  215. 

Digestive  organs,  197 ;  blood  vessels  of,  197. 
Dilution,  rules  for,  7, 13. 
Diphtheria,  109,  111. 
Diplococci,  108. 
Diplococcus   intra   cellularis  meningitidis, 

111. 
Dipping-tube,  94. 
Dirty  paraffin,  43. 
Dirty  sections,  42. 
Dispersion,  139. 
Dissecting  instruments,  1. 
Dissections,  minute,  77. 
Dissociation,  15.  25,  75,  78;  general  rule  for, 

78. 
Dissociators,  formaldehyde,  9,  75 ;  formula© 

for  others,  187. 
Distomes,  220. 
Dogiel,  181. 

Double  staining,  20,  51,  53,  62. 
Doublet  lens,  138. 
Dragon-fly  nymphs,  225 ;  heart  beat  in,  225- 


234 


Animal  Micrology 


Drawing-board,  with  camera  lucida,  147. 

Drawing-table,  Bardeen's  129. 

Drawing  with  camera  lucida,  146, 147. 

Dropping-bottle,  58. 

Dropping  out  of  object  from  paraffin  sec- 
tion, 47. 

Dry  or  dull-looking  areas  in  mounts,  58. 

Duodenum,  198. 

Duval,  orientation  of  young  chick  embryos, 
116. 

Dysentery,  bacillus  of,  111. 

Dityscus,  fore-leg  of,  92. 


Ear,  201. 

Earthworm,  collecting,  222;  coelomic  cor- 
puscles of,  224;  to  immobilize,  224;  to  keep 
alive  in  winter,  224;  nephridia  of,  223; 
ovary  or  testis  of,  224;  setae  of,  224;  sec- 
tioning, 222. 

Eau  de  Javelle,  24. 

Echinoderms,  fertilization  and  early  embry- 
onic stages,  122. 

Eggs,  of  butterfly,  93;  of  chicken,  113;  of 
amphibia,  118;  fish,  hatching-box  for,  123. 

Ehrlich-Biondi  (Heidenhain)  triple  stain, 
174. 

Ehrlich,  hematoxylin,  178 ;  method  for  blood, 
198 ;  methylen  blue  method  for  nerve  tissue, 
180;  triple  stain,  99, 175. 

Elastic  fibers,  184, 185, 193;  fine,  196;  coarse, 
196. 

Embyrograph,  149. 

Embryological  methods,  113. 

Embryology,  of  amphibia,  118;  of  the  chick, 
113;  of  mammals,  119,  120,  121;  of  the 
mouse,  121;  of  the  pig,  120;  of  the  rabbit, 
119;  of  teleosts,  117. 

Embyronic  membranes,  121. 

Embryos,  human,  126;  measuring,  116;  re- 
agents for,  126,  163, 166,  167,  169, 170,  178. 

Encircling  fibers,  196. 
Endothelial  cells,  202. 
Endothelium  of  blood  vessels,  193. 
Eosin,  10,  20,  49,  51,  55,  62,  97, 175  ;  formula,  10. 
Epidermis,  212. 
Epididymus,  210. 
Epistylis,  215. 

Epithelia,  165, 174, 176, 186, 187, 188,  201 ;  isola- 
tion of,  202. 

Epithelium,  ciliated,  201;  columnar  and 
glandular,  201;  cubical,  201;  of  mouth, 
198;  of  small  intestine  and  villi,  198;  of 
stomach,  198;  of  lungs,  211;  of  uriniferous 
tubules,  214;  pigmented,  202;  stratified, 
202;  squamous  or  pavement,  202;  transi- 
tional, 202. 

Erlicki's  solution,  9,  29,  53,  59,  79, 163;  form- 
ula, 9. 

Erythrocytes,  190. 

Erythrosin,  20, 176. 

Esophagus,  198. 

Ether  alcohol,  8,  23,  166. 

Ether  freezing  attachment,  68,  69. 

Eustachian  tube,  201. 


Exner,  123. 

Eycleshymer's  clearer,  22,  62;   methods   of 

orientation,  125. 
Eye,  203;  of  sheep,  162. 
Eyeball,  203;  blood  vessels  of,  203. 
Eyelid,  203. 
Eyepiece,  137. 
Eyepoint,  149, 160. 


Faded  preparations,  restaining,  58. 

Fading  of  blue  color  in  injection  mass,  86. 

Fahrenheit  to  Centigrade  scale,  228. 

Fallopian  tube,  210. 

Farrant's  solution,  22. 

Fasciola,  220. 

Fat,  186, 196. 

Fatigue  of  eyes,  169. 

Feathers,  93. 

Fecundation,  see  fertilization. 

Femoral  artery,  injection  through,  85. 

Fenestrated  membrane,  196. 

Fertilization,  in  Ascaris,  123;  artificial,  122; 

in  amphibia,  echinoderms,  and  teleosts, 

122;  in  mammals,  121. 

Fetuses,  126. 

Fibrillae  in  striated  muscle,  205. 

Fibrillar  (white  fibrous)  connective  tissue, 
197 ;  cells  of,  196. 

Fibrin,  stained  preparation  of,  97. 

Fibrous  tissue,  176. 

Fine  adjustment,  159. 

Fixing,  16,  27,  30;  purpose  of,  16. 

Fixing  agents,  necessary  qualities  of,  16. 

Fixing  and  hardening  agents,  formulae,  161. 

Fixing  sections  to  slide,  23,  41,  64. 

Flagella  of  bacteria,  staining,  111. 

Flat  worms,  90, 170. 

Flatness  of  field,  150. 

Flemming,  161 ;  solution  of,  164, 171, 185, 186. 

Flint  glass,  140. 

Fluid  mounts,  87,  88,  94. 

Flukes,  94. 

Foam  structure,  192. 

Focal  point,  134. 

Focus,  principal,  134;  real,  135;  virtual,  135. 

Formalin,  8,  29,  53,  71,  84,  118,  121, 126;  as  a 
reducing  agent,  166;  with  alcohol  and  ace- 
tic acid,  166. 

Formic  acid,  74, 177. 

Formol-sublimate,  167;  and  acetic  acid,  167. 

Free-hand  sectioning,  33. 

Freezing  method,  67;  carbon  dioxide,  67; 
ether  or  rhigolene,  70. 

Fresh  tissues,  examination  of,  25, 187;  fixing 
and  washing  after  sectioning,  70;  section- 
ing by  the  freezing  method,  69. 

Friable  objects,  sectioning,  43,  44,  63- 

Frog,  embryology  of,  118. 

Fromman,  lines  of,  74. 

Fuchsin,  acid,  20,  176,  189;  and  picric  acid, 
176;  basic,  109,  176. 


Index 


235 


Gabbet's  method  for  tubercle  bacilli,  110. 

Gage,  9,  160,  169;  carbol-xylol  clearer,  9; 
formaldehyde  dissociator,  9. 

Gall  bladder,  198. 

Gait,  31. 

Ganglia,  182,  207 ;  canaliculi  in,  207 ;  periph- 
eral, 73. 

Gastric  glands,  198. 

Gelatin  for  injection  mass,  81. 

General  rules,  6. 

Gentian  violet,  20, 106, 109, 176, 186. 

Germinal  disc  of  chick,  113. 

Gianuzzi,  crescents  of,  198. 

Gilson's  mercuro-nitric  fixing  fluid.  8,  77, 118, 
166;  formula,  8. 

Gilson's  rapid  celloidin  process,  64. 

Gizzard  of  cricket  or  katydid,  77. 

Gland  cells,  162,  165,  175;  of  chironomous 
larva,  192. 

Glanders,  109,  111. 

Glomerulus  of  kidney,  214. 

Glycerin,  21,  22,  87,  88. 

Glycerin  jelly,  21,  22,  79,  89, 90;  formula,  90. 

Glycerin-picrate  mixture,  162. 

Gnat,  91. 

Goblet  cells,  198. 

Gold  chloride,  20,  177;  method  for  nerve- 
endings,  74. 

Gold  size,  87. 

Golgi  method,  71,  72;  mounting  Golgi  prep- 
arations, 72,  73. 

Gonococcus,  111. 

Graafian  follicle,  119,  210. 

Grades  of  alcohol,  7. 

Graduated  cylinder,  4. 

Gram's  solution,  110,  177;  method,  107,  110, 
111. 

Grandry's  corpuscles,  207. 

Grantia,  217. 

Great  water  beetle,  foreleg  of,  92. 

Gregarina,  216. 

Grenacher's  borax-carmine,  9;  see  also  borax- 
carmine. 

Gritty  feeling  in  paraffin  sectioning,  46. 
Grubler  and  Hollborn,  address,  174. 
Gum  and  syrup  mass  for  freezing,  67. 
Gun  cotton,  23. 
Guyer,  101. 


Hair,  212;  development  of,  212;  follicle,  213; 
renewal  of,  213. 

Hanging-drop  preparations,  108. 

Hardening,  15, 17,  27. 

Harder's  glands,  203. 

Hardesty,  72,  73. 

Hard  objects,  to  section,  47,  80. 

Hatching-box  for  fish  eggs,  123. 

Hazy  mounts,  57. 

Heart,  193, 

Heart-beat  in  nymph  of  dragonfly,  225. 

Heidenhain,  10,  52, 174, 178 ;  demilunes  of.  198. 


Hemalum,  84, 177. 

Hematein,  20, 177. 

Hematoidin  crystals,  98. 

Hematoxylin,  19,  20,  162,  165,  168;  ripening 
of,  9,  20,  57 ;  Delafield's,  9,  34,  49,  54,  55,  56, 
57,  68,  84,  89,  91 ;  iron,  10,  52,  53,  55, 123, 178 ; 
Ehrlich's,  178;  Weigert's,  178.      * 

Hematoxylin  and  eosin,  51,  62. 

Hemin  crystals,  98. 

Hemocytometer,  99. 

Hemoglobin  crystals,  97. 

Herbst's  corpuscles,  12. 

Hermann's  fluid,  117, 120,  170, 171, 186;  form- 
ula, 170. 
Hertwig's  macerating  fluid,  76. 
Heterotypical  mitosis,  192. 
His,  116, 129. 

Histological  elements,  isolation  of,  75. 
Homogeneous  immersion  lens,  150, 152. 
Honing  microtome,  44. 
Horn  spoon,  1. 
Huber,  73, 128. 
Human  embryos,  126. 
Hydra,  76,  89,  218;  to  kill  expanded,  89. 
Hydrochloric  acid,  8,  214. 
Hydrofluoric  acid,  25. 
Hydrozoa,  compound,  219. 


Illumination,  150, 159. 

Images,  135 ;  defects  in,  138. 

Imbedding,  22;    paraffin,  37;   celloidin,  59: 

mass  for  well  microtome,  35 ;  a  number  or 

minute  objects,  63;  Ls,  45. 
Immersion  objective,  152. 
Impregnations,  20. 
Incubator,  113. 
Indifferent  fluids,  25, 187. 
Indulin,  190. 
Infiltration,  22. 
Inflammation,  101. 
Influenza,  bacillus  of,  111. 
Infusoria,  168, 173;  quieting,  216. 
Initial  magnifying  power,  144. 
Injected  vessels,  corrosion  of,  86. 
Injecting,  with   a  syringe,  81;   blood   and 

lymph  vessels,  81;    lymphatics,  85;   liver 

fluke,  220;  through  femoral  artery,  85. 
Injection,  test  for  complete,  83;  double,  84 

85;  continuous  air  pressure,  84;   syringe, 

82,  85. 

Injection  masses,  81;  to'keep,  85;  fading  of 
blue  in,  86 ;  cold  fluid  gelatin,  86. 

Injection  methods,  25,  81. 

Ink  for  writing  on  glass,  58. 

Insects,  alimentary  canal  of,  77;  antennae 
of,  93 ;  having  hard  coverings,  93 ;  delicate, 
93;  legs  of,  93;  mounting  entire,  225;  mouth 
parts  of,  77,  93;  muscles  of,  90;  nervous 
system  of,  77;  salivary  glands  of,  77,  192; 
scales  of,  93;  small  or  soft,  93;  stings  of, 
77 ;  wings  of,  93. 

Intercellular  bridges,  202. 
Intercellular  substance,  186. 


236 


Animal  Micrology 


Interstitial  imbedding,  22. 
Intestinal  absorption  of  fat,  198. 
Intestine,  large,  199 ;  small,  199. 
In  toto  preparations,  26,  87. 
Intra  vitam  staining,  173, 179, 183,  216. 
Iodine,  for  washing  after  corrosive  subli- 
mate, 166;  Gram's  solution,  110, 177. 
Iris,  203. 

Iris  diaphragm,  149. 
Iron  hematoxylin,  10,  49, 178. 
Irrigation,  97. 
Isolation  of  histological  elements,  15,  25,  75. 

Jamming  together  of  paraffin  sections,  46. 

Jelly  fish,  219. 

Jelly  of  Wharton,  197. 

Jennings,  215, 222,  224. 

Johnson,  44, 119, 167. 

Joris,  86. 

Julin,  120. 


Kaiserling's  fluid,  31. 

Karyokinesis,  165, 171, 192,  193. 

Keratin,  172, 185. 

Kidney,  blood  vessels  of,  214;  cortex  and 
medulla  of,  214;  glomeruli  of,  214;  injec- 
tion of,  85;  isolation  of  tubules,  214;  me- 
dullary rays  of,  214 ;  nerves  of,  214. 

Killing,  16,  27. 

Kincaid,  31. 

Kleinenberg's  picro-sulphuric,  169. 

Koch-Ehrlich  gentian  violet,  109. 

Kronecker's  fluid,  187. 


Labelling  vessels,  27 ;  slides,  50. 

Labels,  1. 

Lacrymal  glands,  293. 

Landois'  solution,  188. 

Large  intestine,  199. 

Large  objects,  sectioning  in  paraffin,  45. 

Larvae,  transparent,  88;  small  or  soft,  93. 

Larynx,  211. 

Lavdowsky's  mixture,  166. 

Lebrun,  161. 

Lee,  72, 171, 161, 185. 

Leech,  224. 

Length  of  time  for  staining  tissues,  56. 

Lens,  capsule  and  epithelium  of,  203;  fibers, 
*03. 

Lenses,  134;  cleaning,  158;  systems  of,  137. 

Leprosy,  bacillus  of,  110. 

Leptothrix,  108. 

Leucocytes,  191;  feeding,  102;  granules  of, 
190, 191 ;  to  demonstrate  movement  in,  102. 

Ligament,  197. 

Ligamentum  nuchae.  179. 

Light,  for  microscopical  work,  151,  152;  re- 
flected, 150;  transmitted,  150. 

Light  green,  20, 179. 

Lillie,  170. 


Lingual  ribbon  of  snail,  84. 

Lip,  199. 

Lithium  carmine,  107. 

Liver;  amyloid  infiltration  of,  199;  bile 
capillaries  of,  199;  blood  vessels  of,  199- 
cells,  199;  flukes,  220;  hepatic  lobules  of, 
199 ;  injection  of,  85 :  interlobular  connect- 
ive tissue  of,  199. 

Locker,  4. 

Loeffler's  alkaline  methylen  blue,  109;  blood 
serum,  110. 

Logwood,  20. 

Lugol's  solution,  see  Gram's  solution. 

Lung,  212;  blood  vessels  of,  212;  elastic  tis- 
sue of :  212 ;  epithelium  of,  211 ;  foetal,  211 : 
injection  of,  85. 

Lymph,  canals,  183;  capillaries,  193 ;  glands, 
191 ;  spaces,  183. 

Lymphatics,  injection  of,  85. 

Lyons  blue,  10,  20,  49,  51,  179;  formula,  10. 


MacCallum's  macerating  fluid.  188. 
Macerated  tissue,  fixation  of,  78. 
Maceration,  15,  25,  75,  76. 
Magnification,  determination  of,  153, 154. 
Magnifiers,  136. 
Magnifying  power,  144, 152. 
Majenta,  176. 
Malarial  parasite,  103. 
Mallory  and  Wright,  102. 
Mallory's  connective  tissue  stain,  172. 
Mammal-,  maturation,  fertilization,  and  seg- 
mentation in,  121. 

Mammalian    embryos,    early    stages,    119; 

older  stages,  120. 
Mammary  gland,  213. 
Manufacturers,  4. 
Mark,  43. 

Marking  imbedded  specimens,  38. 
Marchi,  207. 
Marrow,  191. 
Mast  cells,  191. 

Material  for  a  course  in  zoology,  215. 
Material,  storing,  30. 
Maturation,  in  amphibia,  echinoderms,  and 

teleosts,  122;  in  Ascaris,  123;  in  mammals, 

121. 
Mayer,  20,  45, 173, 177. 
Mayer's  albumen  fixative,  12,  23;  paracar- 

mine,  184;  hemalum,  177;  carmalum.  173. 
Measurement  of  microscopic  objects,  153. 
Mechanical  stage,  153. 
Medulla  oblongata,  207. 
Medullary  sheath,  270. 
Medullated  fibers,  of  cord  and  tract,  207; 

tracts  of,  178. 
Medusoid  forms,  217. 
Meissner's  corpuscles,  208. 
Membrane  of  Descemet,  204. 
Mercuro-nitric  fixing  fluid,  8, 28,  53,  77 ;  form 

ula,  8. 
Merkel's  fluid,  165. 
Mesothelial  cells,  202. 


Index 


237 


Metagelatin,  90, 

Metallic  substances  for  color  differentia- 
tion, 20,  71. 

Methods,  general  statement  of,  15. 

Methylen  blue,  20, 179;  for  bacteria,  107, 109; 
for-  impregnation  of  epithelia,  183;  for 
nerves  and  nerve-terminations,  180,  181; 
for  non-striated  muscle,  182 ;  for  ordinary 
sections,  182;  immersion  method,  181;  in- 
tra vitam  stain,  180;  Loeffler's  alkaline, 
109;  polychromatic,  180. 

Methyl  green,  20,  76,168.  183,  187;  formula, 
183. 

Methyl  violet,  20, 57;  formula,  183. 

Metric  weights  and  measures,  227. 

Micrococci,  108. 

Micrococcus  tetragenus,  111. 

Micrometer,  stage,  153;  ocular,  155;  filar, 
155. 

Micrometry,  153-55. 

Micron,  44, 155. 

Microscope,  133;  simple,  136;  compound,  137, 
139,  140;  makers,  141-44;  binocular,  146; 
dissecting,  4, 149;  manipulation  of,  157. 

Microscopical  terms  and  appliances,  145. 

Microtome,  well,  35;  for  paraffin,  39,40,60; 
Minot,  39;  Minot-Blake,  40;  oil  for,  44; 
celloidin,  60;  freezing,  67,  68. 

Microtome  knife,  tilt  of,  40;  sharpening,  44. 

Milk,  213. 

Milky  looking  mounts,  57. 

Minot,  116,  121, 174 ;  microtome.  39,  40. 

Minute  dissection,  77,  78. 

Mirror,  155. 

Mites,  88. 

Mitosis,  165, 171,  192,  193;  heterotypical,  192. 

Mollusk,  225. 

Monieza,  220. 

Monocystis,  216. 

Motb,  wings  of,  92. 

Mounting,  22. 

Mouse,  embryology  of,  121, 122. 

Mouth,  epithelium  of,  198. 

Mucin,  172, 199. 

Mucoid  connective  tissue,  197. 

Muller's  fluid,  70, 163,  188,  189;  formula,  163. 

Muscae  volitantes,  155. 

Muscle,  172, 176,  204;  cardiac,  75,187, 188,  204; 
to  tendon,  205;  smooth,  187  ;  voluntary,  75; 
of  insect,  90. 

Muscle  fiber,  branched-striated,  204;  non- 
stria  W,  182,  205 ;  striated,  75,  204;  fibrillae 
of,  205;  end  of  striated,  205;  cardiac,  75. 

Mussel,  gills  of,  225 ;  cross-section  of,  225. 

Myelin,  207. 


Nail,  213. 

Naphthylamin  yellow,  190. 

"Neck  length"  of  embryos,  116. 

Needle,  1. 

Negative  eyepiece,  137. 

Neisser,  method  of  diagnosis  of  diphtheria, 


Nematocysts,  218,  219. 


Nerve,  73;  endings,  74,  177,  180;  tissue,  162, 
163,  176,  180,  186;  degenerated  fibers,  179, 
207 ;  plexuses  in  alimentary  canal,  199 ;  in- 
tra epithelial  fibers,  *07 ;  medullated  fibers, 
207,  208;  non-medullated  fibers,  208;  fiber 
bundles,  208. 

Nerve  cells  and  their  ramifications,  71, 120, 
187. 

Nerve  cells,  Nissl's  method  for,  162, 182. 

Nervous  system,  of  grasshopper,  77 ;  of  ver- 
tebrates, 206. 

Neurokeratin,  208. 

Neuroglia,  73,  208. 

Neutral  red,  183. 

Neutrophil  granules,  175, 191. 

Nitric  acid,  9,  79,  189;  see  also  mercuro- 
nitric  fluid. 

Nissl,  method  for  nerve  cells,  162, 182 :  gran- 
ules, 182, 183,  208, 

Nodes  of  Ranvier,  208. 

Nomenclature  of  objectives  and  oculars,  144. 

Normal  or  indifferent  fluids,  125,  187. 

Normal  saline,  8, 187. 

Nose,  209;  mucous  membrane  of,  209. 

Nosepiece,  139, 158. 

Numerical  aperture,  155. 


Objects  which  will  not  stain,  56. 

Objects  which  alcohol  would  injure,  section- 
ing, 70. 

Objects  of  general  interest,  mounting,  87. 

Objective,  137, 138 ;  immersion,  152, 159 ;  apo- 
chromatic,  146 ;  using  high  power,  158. 

Oblique  illumination,  150. 

Ocular,  137 ;  compensating,  145, 148 ;  search- 
ing, 148 ;  working,  148. 

Odontoblasts,  201. 

Oil,  anilin,  22 ;  of  bergamot,  22;  cedar-wood, 

21,  43,  48,  62 ;  of  cloves,  22,  78 ;  of  origanum, 

22,  62 ;  of  sandal-wood,  22 ;   of  thyme  and 
castor  oil,  62. 

Oil  immersion  lens,  152, 159. 

Olfactory  cells,  209;  nerve  processes  of ,  209. 

Opalina,  216. 

Opaque  mounts,  92. 

Opaque  objects,  150. 

Optical  center  of  lens,  134. 

Optical  principles,  133. 

Orange  G,  20, 183. 

Orcein,  184. 

Organs  and  tissues  with  methods  of  prepara- 
tion, 190. 

Orientation,  38. 

Orienting  chick  embryos,  113. 

Orienting  objects  in  the  imbedding  mass, 
124;  Patton's  method,  125 ;  Eycleshymer's 
methods,  125. 

Orienting  serial  sections,  124. 

Orth's  lithium  carmine,  107. 

Osmic  acid,  18,  78,  79,  116,  120,  167,  168,  183, 
185 ;  discussion  of,  167 ;  vapor,  168. 

Osmic  material,  bleaching,  45. 

Osmium-bichromate  mixture,  72. 

Otoliths,  201. 


238 


Animal  Micrology 


Ova,  210. 
Ovary,  210. 
Over-correction,  157. 
Oviduct,  210. 
Ovogenesis*  210. 
Oxalic  acid,  87, 177. 
Oxyphil  granules,  175. 

Pacinian  corpuscles,  209. 
Pancreas,  199 ;  granules  of,  198. 
Paneth,  cells  of,  198. 
Paper  box  for  paraffin  imbedding,  37. 

Papillae  of  tongue,  200. 

Paracarmine,  169, 184. 

Paraffin,  12,  23;  oven,  12;  dirty,  43. 

Paraffin-asphalt-rubber  method,  44. 

Paraffin  block,  trimming,  39. 

Paraffin  method,  26 ;  imbedding  and  section- 
ing, 37;  staining  and  mounting,  49;  for 
delicate  objects,  54;  compared  with  cel- 
loidiu  method,  63. 

Paramoecium,  215,  217. 

Parfocal,  156. 

Parotid  gland,  199. 

Pathogenic  bacteria,  111. 

Patton,  125. 

Pearl,  167. 

Pectinatella,  222. 

Pedesis,  146. 

Pencil,  glass-marking,  1,  42. 

Penetration,  156. 

Penis,  210. 

Peroxide  of  hydrogen,  9,  24. 

Peyer's  patches,  199. 

Pfluger's  egg  tubes,  210. 

Phloroglucin  method,  189. 

Picric  acid,  18, 168, 184, 189. 

Picric  alcohol,  169. 

Picro-acetic,  117, 122, 123, 169. 

Picro-carmine,  76,  79, 168. 

Picro-hematoxylin,  177. 

Picro-sublimate,  Rabl's  formula,  169;  vom 
Bath's  formula,  169. 

Picro-sulphuric  acid,  117, 121, 169. 

Pig  embryos,  to  obtain  and  prepare,  120, 121 ; 
stages  necessary  for  study,  120;  placenta- 
tion  of,  121 ;  orientation  of,  121. 

Pigment  cells,  193. 

Pigments,  to  remove,  45. 

Placenta,  174,  260. 

Placentation,  121. 

Planaria,  90,  219. 

Platino-aceto-osmic  mixture,  170. 

Plumatella,  222. 

Pneumococcus,  111. 

Polariscope,  156. 

Polypoid  forms,  219. 

Positive  eye-piece,  137. 

Potassium  bichromate,  162. 

Pouring  liquids,  6. 

Preparation  of  reagents,  7. 


Preservation  of  anatomical  specimens,  31. 

Preserving,  18,  30 ;  mixture,  30;  objects  im- 
bedded in  paraffin,  43;  sections  cut  by  the 
freezing  method,  70. 

Prickle  cells,  213. 

Principal  axis  of  lens,  134. 

Protococcus,  94. 

Plotoplasmic  currents,  193. 

Protozoa,  95, 167 ;  cultures,  215 ;  feeding,  216 ; 
permanent  mounted  preparation,  217; 
quieting,  216;  staining,  216. 

Purkinje  cells,  73,  209;  fibers,  194. 

Pyrogallol,  170, 185. 

Pyroxilin,  23. 

Rabbit,  embryology  of,  119 ;  dissection  of,  to 
obtain  embryos,  119,  120;  ova,  time  to  ob- 
tain various  stages,  119. 

Rabl,  169. 

Radula  of  snail,  '94. 

Ranvier,  one-third  alcohol,  188;  picro-car- 
mine, 185 ;  cross  of,  74 ;  nodes  of,  208. 

Rath,  O.  vom,  169. 

Rating  of  objectives  and  oculars,  144. 

Rays  of  light,  133. 

Razor,  section,  1,  33. 

Reagent  bottles,  4. 

Reagents  and  their  preparation,  7;  also 
Appendix  B. 

Reconstruction  of  objects  from  sections,  127 ; 
in  wax,  127 ;  geometrical,  129. 

Reconstruction  points,  125. 

Records,  card,  6,  28;  calendar,  6. 

Rectified  spirit,  13. 

Reduction  division,  193. 

Refraction  of  light,  133. 

Remak's  fibers,  208. 

Removal  of  liquid  from  slide,  55. 

Reproductive  organs,  210. 

Resolving  power,  156. 

Respiratory  organs,  211. 

Restaining  old  mounts,  58. 

Reticular  connective  tissue,  197. 

Retina,  165, 168, 188,  204. 

Reversing  sections,  to  avoid,  40. 

Rhigolene  freezing  attachment,  68,  70. 

Ripart  and  Petit,  164, 187. 

Rolling  of  paraffin  sections,  46. 

Romanowsky  stain,  103. 

Rosin,  183. 

Rotifers,  222. 

Rubbing  sections  off  the  slide,  to  avoid,  56. 

Rubin  S,  176. 

Rules,  general,  6. 

Safranin,  10,  20,165,185;  formula,  185;  and 

gentian  violet,  186. 
Salivary  gland,  199,  200;.  granules  of,  198;  of 

cockroach  or  cricket,  77. 
Salycilic  acid,  170. 
Sarcinae,  108. 
Sarcolemma,  205. 


Index 


239 


Scales  from  wings  of  insects,  93. 

Scalpel,  1. 

Scissors,  1. 

Scheme  for  mounting  whole  objects  or  sec- 
tions, 26. 

Schiefferdecker's  fluid,  188. 

Schneider's  acid  carmine,  174. 

Schultz's  dehydrating  apparatus,  55. 

Schultze's  iodized  serum,  187. 

Sclera,  204. 

Scolex  of  tapeworm,  220. 

Scraping  of  microtome  knife,  47. 

Scratches  across  paraffin  sections,  47. 

Sealing  bottles  and  preparation  jars,  30. 

Sealing  mounts,  89. 

Sebaceous  glands,  213. 

Secondary  axis  of  lens,  134. 

Section  lifter,  1. 

Section  method,  15 ;  simple,  33. 

Sectioning  in  paraffin,  37,  38 ;  in  gum,  67 ;  in 
celloidin,  61 ;  free  hand,  33. 

Sections,  affixing,  23;  drying  of,  55;  milky 
or  hazy,  57;  plane  of,  123,124;  scheme  for 
mounting,  26;  to  stain  by  flooding,  58; 
washing  off  of,  58. 

Semicircular  canals,  201. 
Seminal  vesicle,  210. 
Seminiferous  tubules,  211. 
Serial  sections,  orienting,  123;  see  paraffin 
or  celloidin  method. 

Sharpey,  fibers  of,  195. 

Shell  vials,  support  for,  30. 

Silver,  nitrate,  20, 120, 186;  formula,  186;  for 
nerve,  73. 

Size  of  microscopic  objects,  to  measure,  153. 

Skin  and  its  appendages,  212,  213 ;  blood  ves- 
sels of,  213. 

Slide  box,  1. 

Slides,  1 ;  passing  through  reagents,  56 ;  to 
clean,  57. 

Small  intestine,  198 ;  epithelium  of,  198. 

Small  objects,  to  transfer  through  reagents, 
30;  to  section  free  hand,  34;  to  orient  in 
paraffin,  43. 

Smear  preparations,  blood,  98 ;  bacteria,  105. 

Smegma  bacilli,  110. 

Snail,  225;  or  slug,  84 ;  to  obtain  eggs,  123. 

Sobotta,  122. 

Sodium  chloride,  dissociating  fluid,  188. 

Solutions,  rules  for  making,  6. 

Spawning  of  fish,  122. 

Spectrum,  146;  tertiary,  146. 

Spencer  microscope,  142. 

Spermatogenesis,  211. 

Spermatozoa,  211. 

Spherical  aberration,  138. 

Spicules  of  sponges,  217. 

Spinal  cord,  73,  209. 

Spinal  ganglia,  209. 

Spirillum,  108;  of  Asiatic  cholera,  111. 

Spirogyra,  94. 

Sp©*ges,  217. 


Spores  of  bacteria,  staining,  111. 

Sporozoa,  216. 

Sputum,  to  examine  for  tubercle  bacilli,  110. 

Staining,  19-21,  49;  double  or  multiple,  20, 21, 
51 ;  in  bulk,  54 ;  causes  of  failure  in,  56,  60. 

Staining  jars,  Coplin,  4. 

Stains,  formulae,  170;  classification  of,  19; 
cytoplasmic,  20;  nuclear,  20;  replenishing, 
56;  precise  with  hematoxylin,  56;  for  bac- 
teria, 109. 

Standard  tube-length,  157, 158. 

Staphylococci,  108. 

Staphylococcus  pyogenes  aureus,  111;  al- 
bus,  111. 

Stenders,  4,  49. 

Sting  of  wasp  or  bee,  77. 

Stomach,  200 ;  epithelium  of,  198. 

Stoppers,  to  remove,  13. 

Storing  material,  30. 

Streptococci,  108. 

Streptococcus   pyogenes,    111;    capsulatus, 

Stropping,  45. 
Sublingual  gland,  200. 
Submaxillary  gland,  200. 
Sudan  III,  186. 
Supplies,  1. 
Supporting  tissue,  194. 
Suprarenal  gland,  214. 
Sweat  glands,  213. 
Sympathetic  ganglia,  209. 
Synovial  villi,  197. 
Syphilis,  bacillus  of,  110. 
Syracuse  watch-glass,  4. 
Syringe,  injection,  82,  85. 


Tables  of  equivalent  weights  and  measures, 
227. 

Tactile  corpuscles,  209. 

Tactile  menisci,  209. 

Taenia,  220,  221. 

Tandler,  86. 

Tannic  acid,  97. 

Tapeworm,  94,  220,  221. 

Tastebuds,  200. 

Teasing,  15,  25,  75,  76. 

Teeth  and  other  hard  objects,  grinding,  80. 

Teichmann's  crystals,  98. 

Teleosts,  embryology  of,  117;  to  orient  blas- 
toderms ofj  117;  manipulation  of  embry- 
onic material,  117,  167;  fertilization  and 
early  embryonic  stages,  122. 

Tellyesnicky's  fluid,  163. 

Temperature  of  laboratory,  43. 

Temporary  mounts,  35. 

Tendon,  197;  cells  of,  197;  to  muscle,  197. 

Terminal  bars,  202. 

Testis,  76,  211. 

Tetanus,  bacillus  of,  111. 

Tetracocci,  108. 

Thermometers,  228. 

Thin  sections,  34,  43,  56. 


240 


Animal  Micrology 


Thionin,  20. 

Thymol,  170, 177. 

Thymus  gland,  191,  212. 

Thyroid  gland,  212. 

Tigroid  substance,  182,  208. 

Tilt  of  microtome  knife,  40. 

Tissues  and  organs  with  methods  of  prepa- 
ration, 190. 

Tissues,  killing  and  fixing,  27 ;  which  crum- 
ble in  paraffin,  43;  length  of  time  for 
staining,  56. 

Toluidin  blue,  20. 

Toluol,  22. 

Toisson's  solution,  100. 

Tongue,  200 ;  papillae  and  f olliculi  linguales, 
200. 

Tonsil,  200. 

Tooth,  sectioning  decalcified,  79,  200 ;  devel- 
opment of,  200;  odontoblasts,  201. 

Tough  objects,  sectioning,  47. 

Trachea,  212. 

Tracts  of  medullated  nerve  fibers,  178. 

Transparent  aquatic  organisms,  180. 

Transparent  larvae,  88. 

Trichina,  221,  222;  examining  alive,  222. 

Triplet  lens,  138. 

Tube-length,  157. 

Tubercle  bacilli,  110,  111. 

Turn-table,  4,  88. 

Typhoid,  bacillus  of,  111. 

Umbilicus,  211. 

Under-correction,  157. 

Unna,  182, 184. 

Ureter,  214. 

Urethra,  211,  214. 

Urinary  organs,  213. 

Uriniferous  tubules,  epithelial  cells  of,  214. 

Uterus,  211 ;  and  placentation,  121. 

Vagina,  211. 
Vas  deferens,  211. 
Valves  of  heart,  194. 
Van  Beneden,  120. 
Van  Giesen's  stain,  176. 

Variation  in  thickness  of  paraffin  sections, 
47. 


Vascular  system,  double  injection  of,  84. 

Vein,  194. 

Vertebrata,  226. 

Vials,  4. 

Vision,  conventional  distance  of,  144. 

Visual  purple,  204. 

Volvox,  94. 

Von  Ebner's  fluid,  189. 

Vorticella,  215. 

Vulcanized  fiber  for  celloidin  mounts,  62. 

Walton,  30, 117,  215. 
Wash  bottle,  4. 
Washing,  17. 
Watch-glass,  Syracuse,  4. 
Water  immersion  lens,  152. 
Water  method  for  affixing  sections,  23. 
Water  mites,  88. 

Wax  plates  for  reconstruction,  preparation 
of,  127. 

Weigert,  hematoxylin,  178 ;  method  for  bac- 
teria, 107. 

Weights  and  measures,  table  of  equivalent, 

227. 
Well  microtomes,  35. 
Wharton,  jelly  of,  197. 
White  objects,  orientation  of,  43. 
White  spots  in  paraffin,  38. 
Whitman,  118. 

Whole  objects,  mounting,  26,  87. 
Wire,  copper,  1. 
Wollaston's  camera  lucida,  146. 
Worcester,  119, 167. 
Working  distance,  157. 
Work-table,  157. 
Wright's  stain  for  blood  and  for  malarial 

parasite,  102. 
Wrinkles  in  paraffin  sections,  42,  46. 
Writing  on  glass,  ink  for,  58 ;  pencil  for,  1, 42. 

Xylol,  13,  21,  38,  49;  for  removing  paraffin,  56. 

Zenker's  fluid,  120, 126;   formula,  163. 
Zeiss  microscopes,  141, 144. 
Ziehl-Neelson,  carbol-fuchsin,  109. 
Zoology,  material  for  a  course  in,  215. 


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